Cell preparations for extemporaneous use, useful for healing and rejuvenation in vivo

ABSTRACT

The present invention relates to new plasma or new platelet-rich plasma preparations, new cell dissociation methods, new cell associations or compositions, a method of preparation thereof, a use thereof, devices for the preparation thereof and preparations containing such a platelet-rich plasma preparation and cell associations or compositions. Specifically, the invention provides plasma or platelet-rich plasma alone or in cell composition preparations for use in tissue regeneration and bone regeneration and pain reduction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. application Ser. No.15/605,696, filed May 25, 2017, which is a continuation of U.S.application Ser. No. 15/044,498, filed Feb. 16, 2016, which is acontinuation of U.S. application Ser. No. 14/021,196, filed Sep. 9,2013, now abandoned, which is a continuation of U.S. application Ser.No. 12/438,236, filed Feb. 20, 2009, now U.S. Pat. No. 8,529,957, whichis the U.S. national stage application of International PatentApplication No. PCT/EP2007/058695, filed Aug. 21, 2007, InternationalPatent Application No. PCT/EP2007/058695 claims priority toInternational Patent Application No. PCT/EP2006/065493, filed Aug. 21,2006, the disclosures of which are hereby incorporated by reference intheir entirety, including all figures, tables and amino acid or nucleicacid sequences.

TECHNICAL FIELD

The present invention is related to the field of tissue regeneration,especially skin, cartilage, muscle, tendon, adipose tissue, cornea,peripheral nerves, spine and bone regeneration. It concerns moreparticularly new cell preparations as a biological scaffold, a method ofpreparation thereof, a use thereof, a device for the preparation thereofand preparations containing such cell preparation for extemporaneoususe.

BACKGROUND

The importance of biological autologous materials in the healing processhas been well documented. Most importantly, two biological autologousmaterials have been shown to be directly implicated in the formation ofthe structure of blood clots, which provide a haemostatic barrier whoserole is to ensure hemostasis and seal the wound: (1) fibrin, whichderives from the separation of plasma fibrinogen into two strandsthrough the action of thrombin, and (2) the activated membranes ofplatelets.

The wound healing process is generally presented as the succession of acoagulation phase, an inflammatory process and a regeneration process.

The coagulation phase (blood clotting or clot formation) is a complexprocess whereby a damaged blood vessel wall is covered by a fibrin clotto stop hemorrhage and the repair of the damaged vessel is initiated bythe release in large quantities of cytokines and growth factors fromplatelet alpha granules. The formation of blood clots (formed inphysiological conditions by fibrin, platelets and red blood cells, amongother blood components) is a natural phenomenon that results from tissuetrauma and its role in the wound healing process, as well as in theunion of bone fractures, is well-known.

The inflammation process, which follows the formation of a blood clot,is stimulated by numerous vasoactive mediators and chemotactic factors(specific signals in the form of proteins) released by white blood cellsand platelets. These signals attract macrophages that “clean” the sitefrom bacteria and foreign particles as well as red blood cells beforethe migration of new cells.

The tissue regeneration phase involves the chemoattraction and themitosis of the undifferentiated cells in the scaffold (or growth matrix)formed by the blood clot. The new cells which multiply under thestimulation of platelet growth factors will replace damaged or destroyedcells injured by macrophages.

Growth factors and numerous plasma proteins, also called signalingmolecules, which promote cell migration and division within blood clots,play a crucial role in the wound healing process.

Theoretically, it is possible to amplify the effects of these firstphases in the wound-healing cascade by discarding the red blood cellsand increasing the concentration of growth factors.

Blood clotting amplification can be defined as the formation of an“enriched clot (EC)”. ECs are obtained through the use of plateletconcentrates and have been described in Platelets and Megacaryocytes2004, vol 1 & 2, as “Structure and signals”, Ed. Gibbins andMahaut-Smith, Humana Press, New Jersey.

Platelet-rich plasma (PRP) can be defined as an autologous concentrateof platelets in a small volume of plasma; it has been developed as anautologous biomaterial and has proven to be useful in the heating andregeneration of tissues (Marx et al., 2004, J. Oral Maxillofac. Surg.,62, 489-496). PRP not only consists in a platelet concentrate but alsocontains growth factors (such as platelet-derived growth factor: PDGF,vascular endothelial growth factor: VEGF, transforming growth factor:TGF and epidermal growth factor: EGF) that are actively secreted byplatelets and are known o have a fundamental role in wound healinginitiation process.

For example, PDGF is known to initiate connective tissue healing,including bone regeneration and repair. PDGF also increases mitogenesis(healing cells), angiogenesis (endothelial mitosis into functioningcapillaries) and macrophage activation. VEGF released by the leukocytesis also known to have potent angiogenic, mitogenic and vascularpermeability-enhancing activities on endothelial cells. TGF-β promotescell mitosis and differentiation for connective tissue and bone, acts onmesenchymal stem cells, preosteoblasts and fibroblasts and inhibitsosteoclast formation. EGF is known to induce epithelial development andpromote angiogenesis.

Platelet concentrates are generally used in dental implantology and bonesurgery, notably in the USA. Various techniques of preparation of PRP bycentrifugation processes have been developed. However, due to thesensitivity of the platelet cells and the variability of the efficiencyof the methods of separation of the platelets from the red blood cells,a great variability exist among the methods used for the preparation ofplatelet concentrates (Marx et al., 2004, above; Roukis et al., Adv.Ther., 2006, 23(2):218-37): for example, the laboratory material for invitro diagnostic which is used for platelet preparation, leads to a poorplatelet and other plasma components yield (Marx et al., 2004, above:Anitua 35%, Landsberg 30%, Clinaseal 39%, ACE surgical 33%, Curasan29%). The automated settings from Biomet PCCS & GPS (Marx et al., 2004,above), which not only present the drawback of being a complex processwith prohibitive costs for the process of a blood sample, lead to only ayield of 61% and SmatPreP from Harvest Technology 62%. In those systems,there is obviously an important loss of valuable biologic tissue fromthe patients, therefore there is the need for the development of areliable process collecting the plasma cells with high yields, easy touse and cost effective.

It has been recently demonstrated that the positive effects ofplatelet-rich plasma on bone regeneration spans a limited range ofplatelet concentration and revealed that: an inhibitory effect occurs inthe presence of more than 10⁶ platelets per μl, which is 3 to 4 timesbaseline counts (Weibrich et al., 2004, Bone, 34(4):665-71).

In addition, the obtaining of platelet concentrates still needs the useof relatively complex kits and costly dedicated machinery and theequally costly involvement of specialized technicians. This drawbackmakes the current known methods of preparation of PRP not adapted to apoint-of-care use.

Further, the preparation of cells in view of cellular or tissueregeneration for use in transplantation, post-operative regeneration orfor aesthetic purpose s faced to the long-term conservation problem ofcells and tissues. Tissue or cell cryoconservation is generally used forthe long-term maintaining of tissues or cells, notably platelets, butthis technique has shown serious drawbacks and problems such as crystalformation, osmotic problems, aggregation, inhibition of proteinsynthesis ability, stress protein expression in response to thermalstress, etc. Therefore, tissue or cell cryoconservation is known toalter the cell viability and stability (Agence française de sécuritésanitaire, 2003; Arnaud et al., 1999, Cryobiology, 38, 192-199; Tablinet al., 2001, Cryobiology, 43(2), 114-23). Some of the cryoconservationside effects may be limited by the use of anti-freezing agents such asDMSO or glycerol or other cryopreservatives (US 5, 5891, 617, Oh et al.,Cornea, 26, 840-846) but the concentration of these agents has to beadapted to limit their toxicity and side effects.

Therefore, there is a need for new or alternative method of preparationof cells and tissues suited for use extemporaneously while preservingtheir integrity, notably in terms of growth factors secretion abilityand viability.

SUMMARY

The invention relates to new cell preparation, a method of preparationof new cell preparations, a use of such cell preparations containingsuch as platelet cell preparations, optionally admixed with a cellextract, such as an autologous extract of keratinocytes, bone marrowcells, fibroblasts, periosteum or corneal cells, melanocytes andLangerhans cell; fat cells; muscle cells such as myoblasts and satellitecells; osteoblasts; chondrocytes; umbilical cord cells; Schwann cells orAchilles tendon cells.

The process for the preparation of a platelet concentrate compositionaccording to the invention constitutes a reliable process collecting95%+/−5 of the plasma cells, easy to use and cost effective (Borzini P.et al., in preparation).

In a first aspect, the present invention provides a process for thepreparation of a platelet concentrate composition, comprising the stepsof:

-   a) Centrifuging whole blood in a separator tube selected from:    -   a glass separator tube containing a polyester-based thixotropic        gel and a buffered sodium citrate solution at 0.10 M; and    -   a polyethylene terephthalate separator tube containing a highly        thixotropic gel formed by a polymer mixture and an anhydrous        sodium citrate at 3.5 mg/mL;-   b) Separating the enriched platelet rich plasma from the full plasma    by removing half of the supernatant containing the platelet poor    plasma;-   c) Re-suspending the enriched plasma;-   wherein the centrifugation step is performed at a force of or about    1500 g up to about 2000 g in a sufficient length of time to form a    barrier between the plasma containing the platelets, the lymphocytes    and the monocytes and the pellet containing the erythrocytes; the    separation step b) is made by collecting the supernatant from atop    of said barrier and wherein the enriched plasma is enriched in    leucocytes, thrombocytes and adhesion proteins (for example,    fibronectin) as compared to native whole blood.

In a second aspect, the present invention provides an isolated plateletconcentrate composition comprising:

-   a) plasma;-   b) platelets at a concentration of at least 300×10⁹ cells/L;-   c) white blood cells at a concentration of at least 7.0×10⁹ cells/L;-   d) fibrinogen at a concentration of at least 3 mg/L;-   and wherein the erythrocyte concentration is less than 0.6×10¹²    cells/L.

In a third aspect, the present invention provides a wound healantcomposition comprising:

-   a) plasma;-   b) platelets at a concentration of at least 300×10⁹ cells/L;-   c) white blood cells at a concentration of at least 7.0×10⁹ cells/L;-   d) fibrinogen at a concentration of at least 3 mg/L;-   e) coagulation activator in a vol. ratio (platelet    concentrate:coagulation activator) of about 10:1 to about 10:3;-   f) optionally an autologous cell extract, such as an extract of    keratinocytes, bone marrow cells, osteoblasts; chondrocytes,    fibroblasts, periosteum or corneal cells, melanocytes and Langerhans    cell; fat cells; muscle cells such as myoblasts and satellite cells;    umbilical cord cells; Schwann cells, tendon cells or pancreas islet    cells;-   and wherein the erythrocyte concentration is less than 0.6×10¹²    cells/L.

In a fourth aspect, the present invention provides a process for thepreparation of a wound healant composition comprising:

-   a) Providing a platelet concentrate of the invention;-   b) Admixing the platelet concentrate with a coagulation activator in    a vol. ratio (platelet concentrate:coagulation activator) of about    10:1 up to about 10:3;-   c) Optionally admixing autologous cell extract, such as extract of    keratinocytes, bone marrow, fibroblasts, periosteum or corneal    cells, melanocytes and Langerhans cell; fat cells; muscle cells such    as myoblasts and satellite cells; osteoblasts; chondrocytes;    umbilical cord cells; Schwann cells or Achilles tendon cells.

In a fifth aspect, the present invention provides a device for thepreparation of a platelet concentrate from whole blood comprising aseparator tube wherein the separator tube is selected from:

-   a glass separator tube containing a polyester-based thixotropic gel    and a buffered sodium citrate solution at 0.10 M; and-   a polyethylene terephthalate separator tube containing a highly    thixotropic gel formed by a polymer mixture and an anhydrous sodium    citrate at 3.5 mg/mL;-   characterised in that the device has an inlet for introducing said    whole blood, is held in a vacuum intended to aspirate the whole    blood sample, is sterile, has a usable vacuum of or about 8 to about    10 mL and is suitable for undergoing centrifugation.

In a sixth aspect, the present invention provides a use of a plateletconcentrate according to the invention for the manufacture of amedicament for healing of wounds or for promoting bone or periodontumgrowth and/or bone and/or tissue regeneration.

In a seventh aspect, the present invention provides a use of a plateletconcentrate according to the invention for the manufacture of a cosmeticpreparation for use as anti-aging agent or skin repairing agent such asa scar repairing agent, a wrinkle filling and/or repairing agent.

In an eighth aspect, the present invention provides a pharmaceuticalcomposition comprising platelet concentrate according to the inventionand a pharmaceutically acceptable carrier.

In a ninth aspect, the present invention provides a cosmetic compositioncomprising platelet concentrate according to the invention and acosmetically acceptable carrier.

In a tenth aspect, the present invention provides an implantable devicefor use in tissue regeneration therapy comprising:

-   (a) a permeable core comprising a platelet concentrate of the    invention; and-   (b) an external jacket surrounding said core, said jacket comprising    a biocompatible material, preferably bioresorbable.

In an eleventh aspect, the invention provides a kit adapted for tissueregeneration comprising a separator tube according to the invention,phlebotomy accessories for the preparation of the wound healantaccording to the invention and an applicator device (e.g. a doublesyringe) for the simultaneous dispensation onto the wound of theplatelet concentrate according to the invention and a coagulationactivator.

In a twelfth aspect, the invention provides a method for promoting woundhealing and/or sealing and/or tissue and/or bone regeneration in a woundof a human or a lower animal comprising:

-   a) Providing a wound healant according to the invention;-   b) Applying a therapeutically effective amount of the said wound    healant to a wound, a damaged tissue or a damaged bone.

In a thirteenth aspect, the invention provides a method for inducingperiodontal regeneration in a wound or a periodontal defect of a mammalwith periodontal disease or other condition requiring periodontalregeneration comprising:

-   a) Providing a wound healant according to the invention;-   b) Applying a therapeutically effective amount of the said wound    healant to the said wound or said periodontal defect or cavity;-   c) Optionally inserting a periodontal barrier, wherein the barrier    is positioned between the gingival tissue and the wound treated    according to steps a) and b) and the said barrier is selected from a    membrane, a biodegradable polymer and/or a biocompatible porous    material;-   d) Closing the wound.

In a fourteenth aspect, the invention provides a method for promotingskin regeneration in a scar or a wrinkle from human or lower animalcomprising:

-   a) Providing a wound healant according to the invention;-   b) Filling the skin scar or wrinkle line with the said wound    healant.

In a fifteenth aspect, the present invention provides a process for thepreparation of a cell composition, comprising the steps of:

-   (a) Centrifuging whole blood in a separator tube selected from:    -   a glass separator tube containing a polyester-based thixotropic        gel and a buffered sodium citrate solution at 0.10 M; and    -   a polyethylene terephthalate separator tube containing a highly        thixotropic gel formed by a polymer mixture and an anhydrous        sodium citrate at 3.5 mg/mL;-   (b) Optionally separating the enriched platelet rich plasma from the    full plasma by removing half of the supernatant containing the    platelet poor plasma;-   (c) Re-suspending the enriched plasma;-   (d) Providing a cell extract such as an extract of dermal cells such    as keratinocytes, fibroblasts, melanocytes and Langerhans cell; fat    cells; bone marrow cells; muscle cells such as myoblasts and    satellite cells; osteoblasts; chondrocytes; periosteal membrane    cells; corneal cells; umbilical cord cells; Schwann cells, tendon    cells or pancreas islet cells;-   (e) Admixing the platelet concentrate obtained under step (c) with    the cell extract obtained in (d);-   wherein the centrifugation step a) is performed at: a force of or    about 1500 g up to about 2000 g in a sufficient length of time to    form a barrier between the plasma containing the platelets, the    lymphocytes and the monocytes and the pellet containing the    erythrocytes; the separation step b) is made by collecting the    supernatant from atop of said barrier and wherein the enriched    plasma is enriched in leucocytes, thrombocytes and adhesion proteins    (for example, fibronectin) as compared to native whole blood.

In a sixteenth aspect, the present invention provides a process for thepreparation of a wound or tissue healing composition, comprising thesteps of:

-   a) Centrifuging whole blood in a separator tube selected from:    -   a glass separator tube containing a polyester-based thixotropic        gel and a buffered sodium citrate solution at 0.10 M; and    -   a polyethylene terephthalate separator tube containing a highly        thixotropic gel formed by a polymer mixture and an anhydrous        sodium citrate at 3.5 mg/mL;-   b) Optionally separating the enriched platelet rich plasma from the    full plasma by removing half of the supernatant containing the    platelet poor plasma;-   c) Re-suspending the enriched plasma;-   d) Admixing the platelet concentrate obtained under step (c) with a    coagulation activator in a vol. ratio (platelet    concentrate:coagulation activator) of about 10:1 up to about 10:3;-   e) Providing a cell extract such as an extract of dermal cells such    as keratinocytes, fibroblasts, melanocytes and Langerhans cell; fat    cells; bone marrow cells; muscle cells such as myoblasts and    satellite cells; osteoblasts; chondrocytes; periosteal membrane    cells; corneal cells; umbilical cord cells; Schwann cells, tendon    cells or pancreas islet cells;-   f) Admixing the platelet concentrate admixture obtained under    step (d) with the cell extract obtained in (e); wherein the    centrifugation step is performed at a force of or about 1500 g up to    about 2000 g in a sufficient length of time to form a barrier    between the plasma containing the platelets, the lymphocytes and the    monocytes and the pellet containing the erythrocytes; the separation    step b) is made by collecting the supernatant from atop of said    barrier and wherein the enriched plasma is enriched in leucocytes,    thrombocytes and adhesion proteins (for example, fibronectin) as    compared to native whole blood.

In a seventeenth aspect, the present invention provides an isolated cellcomposition comprising:

-   a) plasma;-   b) platelets at a concentration of at least 300×10⁹ cells/L;-   c) white blood cells at a concentration of at least 7.0×10⁹ cells/L;-   d) fibrinogen at a concentration of at least 3 mg/L;-   e) a cell extract, such as an extract of dermal cells such as    keratinocytes, fibroblasts, melanocytes and Langerhans cell; fat    cells; bone marrow cells; muscle cells such as myoblasts and    satellite cells; osteoblasts; chondrocytes; periosteal membrane    cells; corneal cells; umbilical cord cells; Schwann cells, tendon    cells or pancreas islet cells wherein cells are at a concentration    of about 10⁵ to about 10⁶ cells/L or a concentration of about 10⁵ to    about 10⁶ cells/ml of plasma or enriched plasma; and wherein the    erythrocyte concentration is less than 0.6×10¹² cells/L.

In an eighteenth aspect, the present invention provides an isolated cellcomposition comprising:

-   a) plasma;-   b) platelets at a concentration of at least 300×10⁹ cells/L;-   c) white blood cells at a concentration of at least 7.0×10⁹ cells/L;-   d) fibrinogen at a concentration of at least 3 mg/L;-   e) coagulation activator in a vol. ratio (platelet    concentrate:coagulation activator) of about 10:1 to about 10:3;-   f) a cell extract, such as an extract of dermal cells such as    keratinocytes, fibroblasts, melanocytes and Langerhans cell; fat    cells; bone marrow cells; muscle cells such as myoblasts and    satellite cells; osteoblasts; chondrocytes; periosteal membrane    cells; corneal cells; umbilical cord cells; Schwann cells, tendon    cells or pancreas islet cells wherein cells are at a concentration    of about 10⁵ to about 10⁶ cells/L or at a concentration of about 10⁵    to about 10⁶ cells/ml of plasma or enriched plasma;-   and wherein the erythrocyte concentration is less than 0.6×10¹²    cells/L.

In a nineteenth aspect, the invention provides a wound or tissue healingcomposition comprising an isolated cell composition according to theinvention.

In a twentieth aspect, the invention provides a method for promotingwound healing and/or sealing and/or regeneration of a tissue and/or acartilage and/or a bone and/or a nerve in a human or a lower animalcomprising:

-   a) Providing a wound or tissue healing composition or a cell    composition according to the invention;-   b) Applying a therapeutically effective amount of the said a wound    or tissue healing composition or cell composition to a wound, a    damaged tissue or a damaged cartilage or a damaged bone.

In a twenty-first aspect, the invention provides a method for increasingadipose tissue volume in a mammal with a dermal fat graft or othercondition requiring adipose tissue regeneration comprising:

-   a) Providing a fat cell composition according to the invention;-   b) Applying a therapeutically or cosmetically effective amount of    the said fat cell composition to the dermal fat graft or the adipose    tissue requiring adipose tissue regeneration;-   c) Optionally inserting a surgical flap or implant, wherein the    surgical flap or implant, is positioned in the site requiring    regeneration or volumetric amplification and the said surgical flap    or implant comprises a combination of a fat cell preparation    according to the invention and plasma or enriched plasma material.

In a twenty-second aspect, the invention provides a method for inducingmyocardial regeneration in a mammal with myocardial deficiency or othercondition requiring myocardial regeneration tissue regenerationcomprising:

-   a) Providing a muscle cell or a bone marrow cell composition    according to the invention;-   b) Applying a therapeutically effective amount of the said muscle    cell composition to the myocardial tissue requiring regeneration.

In a twenty-third aspect, the invention provides a method for inducingcorneal regeneration in a mammal with corneal deficiency or othercondition requiring corneal regeneration comprising:

-   a) Providing a cornea cell composition according to the invention;-   b) Applying a therapeutically effective amount of the said corneal    cell composition to the corneal tissue requiring regeneration.

In a twenty-fourth aspect, the invention provides a method for inducingarticular or cartilage regeneration in a mammal with articular orcartilage deficiency or other condition requiring articular or cartilagetissue regeneration comprising:

-   a) Providing a chondrocyte cell or bone marrow cell composition    according to the invention;-   b) Applying a therapeutically effective amount of the said    chondrocyte cell composition to the articular or cartilage tissue    requiring regeneration;-   c) Optionally inserting a surgical flap or implant, wherein the    surgical flap or implant, is positioned in the defect of the    cartilage or under a periosteal patch, and the said surgical flap or    implant comprises a combination of a chondrocyte or bone marrow cell    composition according to the invention and plasma or enriched plasma    material.

In a twenty-fifth aspect, the invention provides a method for promotingskin regeneration in a scar, a wrinkle or a fat deficiency from human orlower animal comprising:

-   a) Providing a wound or tissue healant or a cell composition    according to the invention;-   b) Filling the skin scar, wrinkle line or fat deficiency with the    said wound or tissue healant or cell composition according to the    invention.

In a twenty-sixth aspect, the invention provides a method for inducingperipheral nerve regeneration in a mammal with peripheral nerve damage,nerve suture or spinal cord injury or other condition requiringperipheral nerve regeneration comprising:

-   a) Providing a Schwann cell composition according to the invention;-   b) Applying a therapeutically effective amount of the said Schwann    cell composition to the peripheral nerve requiring regeneration.

In a twenty-seventh aspect, the invention provides a method for inducingbone regeneration in a mammal with bone damage, bone deficiency or othercondition requiring bone regeneration comprising:

-   a) Providing a bone marrow cell or osteoblast cell composition    according to the invention;-   b) Applying a therapeutically effective amount of the said bone    marrow cell or osteoblast cell composition to the bone requiring    regeneration.

In a twenty-eighth aspect, the invention provides a method for thetreatment of type I diabetes, insulin-dependent diabetes orhyperglycaemia in a mammal comprising:

-   a) Providing a pancreas islet cell composition according to the    invention;-   b) Applying a therapeutically effective amount of the said pancreas    islet cell composition to the patient, for example by injection.

In a twenty-ninth aspect, the invention provides a method for thetreatment of urinary incontinence in a mammal or other conditionrequiring bladder regeneration comprising:

-   a) Providing a myoblast cell composition according to the invention;-   b) Applying a therapeutically effective amount of the said myoblast    cell composition to the bladder neck requiring regeneration.

In a thirtieth aspect, the invention provides a method for the treatmentof anal incontinence in a mammal or other condition requiring analmuscle regeneration comprising:

-   a) Providing a myoblast cell composition according to the invention;-   b) Applying a therapeutically effective amount of the said myoblast    cell composition to the para-anal area requiring regeneration.

In a thirty-first aspect, the invention provides a method for thetreatment of reflux oesophagitis or gastro-oesophageal reflux disordersin a mammal or other condition requiring oesophageal sphincterregeneration comprising:

-   a) Providing a myoblast cell composition according to the invention;-   b) Applying a therapeutically effective amount of the said myoblast    cell composition to the oesophageal sphincter requiring    regeneration.

In a thirty-second aspect, the present invention provides a use of acell preparation according to the invention for the manufacture of amedicament for healing of wounds or tissues or for promoting bone orperiodontum growth and/or bone and/or tissue regeneration such as skin,cartilage, muscle, tendon, adipose tissue, cornea, peripheral nerves,spine or bone regeneration.

In a thirty-third aspect, the present invention provides a use of a cellcomposition according to the invention for the manufacture of a cosmeticpreparation for use as anti-aging agent or skin repairing agent such asscar repairing agent, lipoatrophy repairing agent or wrinkle fillingand/or repairing agent.

In a thirty-fourth aspect, the present invention provides apharmaceutical composition comprising a cell composition according tothe invention and a pharmaceutically acceptable carrier.

In a thirty-fifth aspect, the present invention provides a cosmeticcomposition comprising cell composition according to the invention and acosmetically acceptable carrier.

In a thirty-sixth aspect, the present invention provides an implantabledevice for use in tissue regeneration therapy comprising:

-   a) a permeable core comprising a cell composition of the invention;    and-   b) an external jacket surrounding said core, said jacket comprising    a biocompatible material, preferably bioresorbable.

The uses, methods and compositions according to the invention are usefulin the regeneration and/or rejuvenation of tissues, bones and/orcartilages. The uses, methods and compositions according to theinvention are particularly useful in the treatment of diabeticneuropathic ulcers or decubitus sores; bone and cartilage damages suchas deep joint cartilage or chondral damages such as surgical repair oftorn tendons; arthritis in joint caused by traumas or by aging; rotatorcuff disorders; non-healing wounds such as vasculitis induced wounds,for example in lower equine limb; periodontal diseases; implant surgery;cardiac muscle damages such as in chronic cardiac failure, heartfailure, ischemic and non-ischemic disorders, cardiomyopathy;gastro-oesophageal reflux disease; anal or urinary incontinence; facialsurgery such as facial surgery induced alopecia (alopecia due to hairfollicle loss in the side burn areas), face-lift surgery (rhytidectomy),rhinoplasty, dermal fat grafts (in the treatment of facial augmentation,congenital hemiatrophy of the face such as congenital cartilage noseatrophy and lipoatrophy such as in HIV/AIDS suffering patients, erosionand arthroscopy); wound healing complications such as after eyelidblepharoplasty; conical disorders such as corneal opacity such as thosecaused by chemical burns, affliction by Steven's Johnson syndrome andcorneal ulcers; scarring of the cornea; dry eye syndrome; haematologicaldiseases such as Thalassaemia; peripheral nerve damage, nerve suture andspinal cord injury; bone defects or disorders such as bone graft or bonefracture, skin damages or disorders such as acne (especially afterdermabrasion treatment), burns, rubella or small pox scars, vitiligo,lipoatrophy, Kaposi's sarcoma, skin keloids or Dupuytren's palmarfibromatosis.

In another aspect, the uses, methods and compositions according to theinvention are useful in the regeneration and/or rejuvenation of skintissues, particularly in promoting and/or initiating skin regenerationsuch as reducing skin wrinkles, acne (especially after dermabrasiontreatment), burns, rubella or small pox scars, vitiligo and lipoatrophy(e.g. anti-aging compositions and skin regeneration compositions),amelioration of nasolabial lines and treatment of skin damages ordisorders such as skin burns, Kaposi's sarcoma, skin skeloids orDupuytren's palmar fibromatosis and in the reduction of pain associatedwith skin and tissue regeneration.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of the variation of concentrationin growth factors (PDGF-AB, EGF and VEGF) of a platelet concentratecomposition according to the invention versus time (T in hours) afterthe centrifugation step in the preparation process of the invention.

FIG. 2 is a schematic representation of the outcome of the treatment ofa skin graft donor site with a preparation containing a plateletconcentrate composition according to the invention in comparison with acontrol group in terms of healing time in days (HT), pain at day 5 on ascale 0 to 10 (P) and epithelization at day 5 on a scale 0 to 7 (E).Control group: C, platelet-rich preparation alone: RegenPRP™,platelet-rich preparation and autologous keratinocytes: RegenExtracell™.The dotted line indicates when the first bandage is changed at day 5.

FIG. 3 represents the morphology of a human fibroblast expanded in cellpreparation according to the invention under the condition described inExample 7, showing branching and filopodia ×5′000 (Olympus® invertedmicroscope).

FIG. 4a represents a 3D scaffold of human fibroblasts from a cellpreparation according to the invention under the condition described inExample 7.

FIG. 4b represents monolayers of fibroblasts densely packed in 3Dculture from a cell preparation according to the invention under thecondition described in Example 7.

DETAILED DESCRIPTION

The following paragraphs provide definitions of the terms according tothe invention and are intended to apply uniformly throughout thespecification and claims unless an otherwise expressly set outdefinition provides a broader definition.

By the expression “thixotropic” is meant a gel that becomes more fluidas a result of agitation or pressure, i.e. a gel which viscosity isdecreasing as a result of agitation or pressure. The term viscosityrefers to those characteristics of the specified material(s) determiningthe degree of gelation, such as for example the firmness or hardness ofthe material, the degree to which the material resists flowing like afluid. A thixotropic gel according to the invention comprising apolyester gel or a mixture thereof which is water insoluble andchemically inert to blood constituents which can be used in accordancewith the invention. Typical thixotropic gels are used in blood cellsseparation for diagnostics and proteomics purposes.

By the expression “point-of-care” is meant all services provided topatients at the bedside.

By the expression “phlebotomy accessories” or “venipuncture accessories”is meant accessories that allow the puncture of a vein with a needle forthe purpose of drawing blood.

By the expression “wound healant” or “wound sealant” or a “tissuehealing composition” is meant an agent or a composition that is able topromote and/or increase the speed and/or quality of cicatrisation of awound. Wound healants or sealants are able to promote tissueregeneration.

By the expression “wound” is meant any damaged tissue, for examplefollowing trauma or surgery. Wounds in mammals, include for examples bedsores, ulcers, lacerations and burns, graft sites (graft donor andacceptor sites), fistulas, periodontal tissue damages, diabeticnon-healing wounds, consequences of traumas or any surgery act. In itsgeneral sense the expression is intended to also encompass skin damageswhere the skin surface presents some depression without necessarily acut on its surface such as age-related tissue damages (e.g. wrinkles)and scars such as for example acne (especially after dermabrasiontreatment) or rubella scars.

By the expression “PRP” is intended to mean a platelet-rich-plasma,preferably of human origin, more preferably autologous, prepared by theprocess of the invention in order to pellet and remove erythrocytes andconcentrate the plasma in leucocytes, thrombocytes and adhesion proteinsas compared to native whole blood.

By the expression “autologous” or “autogenic” or “autogenous” isintended a method of the invention using a single donor's blood andwherein the blood extracted from this donor is intended for use on thesame donor. As opposed, “allogeneic” methods are using blood from one ormore third patties for use on a donor (“homologous” or “heterologous”).An autologous product avoids some of the common problems associated withthe use of biological materials from third parties, such as for examplescreening to assure that the donor was biologically or immunologicallycompatible with the patient and potential contamination with hepatitis,HIV, prion, Creutzfeldt-Jacob disease and the like.

By the expression “coagulation activator” is intended an agent that isable to trigger or activate coagulation. A coagulation activatorcomprises a thrombin activator and/or a fibrinogen activator.

By the expression “thrombin activator” is intended an agent that is ableto activate thrombin and to trigger coagulation. Typical thrombinactivators are certain cofactors such as sodium or calcium. Inpracticing this invention, thrombin activation preferably occurs in thepresence of calcium ions. Calcium ions are generally added to theplatelet concentrate as a salt solution to provide a final concentrationgenerally of or about 0.1 mg/mL of platelet concentrate. Suitablecalcium salts include, without limitation, CaCO₃, and CaSO₄. A preferredcalcium salt for use in the invention is calcium chloride (CaCl₂). CaCl₂is available as calcium chloride injection, USP 10% (Regen Lab,Switzerland).

By the expression “fibrinogen activator” is intended an agent that isable to activate the conversion of fibrinogen into fibrin and triggersthe formation of the clot. Typical fibrinogen activators are thrombin orbatroxobin. The term thrombin may include calcified thrombin, inparticular, from or about 100 to about 10 units of thrombin per 1 mL of10% of aqueous calcium chloride solution; it may include calcifiedbovine thrombin, allogeneic thrombin or recombinant human thrombin,preferably autologous thrombin. A fibrinogen activator can be anenriched thrombin composition such as thrombin compositions as describedin U.S. Pat. No. 6,472,162 or an autologous thrombin serum according tothe invention.

By the expression “therapeutically effective amount” is intended theamount or amounts of the constituent elements or combination thereofnecessary to enhance wound healing such as, for example, the reductionin the volume or surface area of a wound, the increase in the amount ofgranulation tissue or other biological material facilitating collagenlay down, vascular in growth, fibroblast proliferation or overallhealing; All of the versions of the invention described herein areassumed to have the therapeutically effect amount(s) of constituentsubstances, or combinations thereof.

By the expression “pharmaceutically acceptable carrier” is intendedpharmaceutically acceptable additional ingredient such as stabilizers,antimicrobial agents, buffers, adjuvants, anaesthetics, corticosteroidsand the like.

By the expression “cosmetically acceptable carrier” is intendedcosmetically acceptable additional ingredient such as stabilizers,buffers, colouring agents, flavouring agents, adjuvants, and the like.

The compositions, uses and methods according to the invention areparticularly useful in wound or tissue healing or regenerationtreatments, especially the treatment of traumatic or surgical woundssuch in the fitting and/or holding and/or sealing of native orprosthetic grafts (especially skin, bone grafts and/or dental prosthesesor implants or the like, including also the graft donor site); treatmentof vasculitis; ulcers such as diabetic neuropathic ulcers or decubitussores; radiodermatitis (e.g. after irradiation on an epidermoidal skincarcinoma) and closing fistulas (such as for cyclists).

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of cardiac disorders, cardiacregeneration such as in the treatment of heart failure, chronic cardiacfailure, ischemic and non-ischemic cardiac failure and cardiomyopathy.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of urinary and/or analincontinence.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of reflux oesophagitis and/orgastro-oesophageal reflux disorder.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of skin damages such as inskins damaged by radiations (radiodermatitis or sun damaged skin), agedskins or burned skins and/or in the amelioration of facial wrinkles,rhytids, acne (especially after dermabrasion treatment), burns, rubellaor small pox scars, vitiligo, lipoatrophy or lipodystrophy, Kaposi'ssarcoma, skin skeloids or Dupuytren's palmar fibromatosis and/or in skinrejuvenation treatments.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of lipoatrophy such as inHIV/AIDS patients and in other congenital hemiatrophy of the face suchas congenital cartilage nose atrophy.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of bone, cartilage andarticular disorders such as chondral damage, arthritis, cartilage and/orbone injury such as deep cartilage damage and/or erosion and/orarthroscopy, tendon torn and rotator cuff in shoulder.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of hematological diseases suchas Thalassaemia.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of corneal disorders such asdry eye syndrome; corneal opacity such as those caused by chemicalburns, affliction by Steven's Johnson syndrome; scarring of the corneaand corneal ulcers.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of peripheral nerve damage,nerve suture and spinal cord injury.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of type1 diabetes,insulin-dependent diabetes and/or hyperglycaemia.

Further, the compositions, uses and methods according to the inventionare particularly useful in the treatment of bone defects or disorderssuch as bone graft or bone fracture.

The use of the resulting composition the invention can be furthermodified before application and according to the therapeutic objective.

Compositions of the invention can be used together with bone fillingmaterials, especially resorbable filling materials such ashydroxyapatite (calcium phosphate ceramic used as a biomaterial) ordemineralised bone, or used as a mixture with bone extracts in a processfor the regrowth of bone for example in craniofacial and orthopaedicprocedures.

Compositions of the inventions may be used as a wound sealant in plasticsurgery including burn grafting and other free skin graft applications,for example in oncology for favouring tissue regeneration, includingspeeding (neo)vascularization.

The compositions according to the invention are particularly useful inwound healing treatments at the skin graft donor site. The removal of askin graft on a healthy skin creates a new wound at the donor's sitewhich normally heals spontaneously between 12 to 14 days. However, thiscicatrisation is extremely demanding for the body, especially if thedonor site is broad or the person is less resistant (e.g. burn victims,people suffering from multiple traumas, people treated with corticoids,children or elderly) and the energetic losses are even increased by theloss in minerals, trace elements and proteins induced by the fluidlosses from the new wound. In addition, important pain during the first8 days is often present on the graft donor's site. Pain reductiontreatments are often used such as the use of analgesics (e.g. morphine)and/or hydrocellular wound dressings, however pain remains present,especially during the dressing change that occurs imperatively within 48hours up to 1 week after the graft removal. In addition, thehydrocellular wound dressings have the drawbacks not only to be ratherexpensive but also by maintaining humidity on the wound, to prevent itsdrying, to increase the wound deepness, to favour the outbreak ofbacterial infections and to lead to non-esthetic scars. Therefore, astimulation of the skin graft donor site healing is very desirable.

Compositions of the invention are particularly adapted to chronic woundsthat may lack sufficient blood circulation to facilitate the woundhealing cascade.

The compositions and methods according to the invention may be also usedin the treatment of periodontal disease where a loss and/or a damage ofthe periodontal tissues is observed, such a treatment comprising forexample placing at the periodontal site or cavity in a human or a loweranimal in need of periodontal tissue regeneration a compositionaccording to the invention.

The compositions according to this invention are effective ineliminating or greatly reducing post-operative bleeding andextravasation or loss of serous or other fluid in these applications, inreducing the infection risk caused by most bacteria and/or enhancesconnective tissue formation compared to natural healing (i.e. noexogenous agents added) or to healing obtained through the use of otherplatelet concentrates prepared with known methods.

The compositions according to the invention are particularly useful inthe preparation of pharmaceutical for promoting and/or initiating woundhealing and/or tissue regeneration or for the preparation of cosmeticcompositions for skin regeneration such as reducing skin wrinkles, acne(especially after dermabrasion treatment), rubella or small pox scars,vitiligo and lipoatrophy (e.g. anti-aging compositions and skinregeneration compositions).

The compositions of the present invention may be administered locally orinjected in the wound or in or near to the grafted organ or injectedsubcutaneously. Local administration may be by injection at the site ofinjury or defect or by insertion or attachment of a solid carrier at thesite, or by admixture with a cream or emulsion, or by inclusion in atissue or paper or hydrogel carder, or by direct, topical application ofthe composition of the invention such as in the form of eye drops.Preferably, the compositions are readily syringable compositions. Themode of administration, the dosage administered, as single or multipledoses, to an individual will vary depending upon a variety of factors,including pharmacokinetic properties, patient conditions andcharacteristics (sex, age, body weight, health, size), extent ofsymptoms, concurrent treatments, frequency of treatment and the effectdesired.

The compositions of the present invention may be administered incombination with a co-agent useful in the treatment of tissueregeneration such as a healing agent, a wrinkle filler, an anti-agingagent such as an anti-aging vitamin complex, an antibacterial agent,antibiotic agent, an corticosteroid agent, an antalgic and analgesicagent, or an anesthetic agent like adrenaline, etc. . . . The inventioncomprises compositions combined with a co-agent useful in the treatmentof tissue regeneration for simultaneous, separate or sequential use intissue regeneration therapy such as wound healing, bone and periodontumgrowth repair.

The compositions of the invention, the device and procedures for thepreparation of autologous platelet concentrates or cell compositions ofthe invention are particularly useful for therapeutic use, particularlyas autogenous biological glue in a haemostatic system intended toaccelerate the physiological process of tissue regeneration, for examplein dental implantology, skin and bone surgery, cartilage and tendonsurgery, corneal and peripheral nerve regeneration and cardiac surgery.The compositions of the invention, the device and procedures for thepreparation of autologous platelet concentrates and cell composition ofthe invention are particularly useful for cosmetic use, particularly asautogenous rejuvenation material intended to be used for example aswrinkle, scar or fat deficiency filler, alone on in combination with atleast one anti-aging agent.

The platelet concentrate of the invention may be combined with anautologous cell extract preparation such as for example keratinocytes,bone marrow cells, osteoblasts, chondrocytes, fibroblasts, periosteum,melanocytes and Langerhan's cell; fat cells; bone marrow cells; musclecells such as myoblasts and satellite cells; periosteal membrane cells;corneal cells; umbilical cord cells; tendon cells or pancreatic isletcells, Keratinocytes can be harvested through a method described byReinwald and Green, 1975, Cell, 6(3):331-43. Other mentioned cells canbe harvested through methods described in “Culture de cellules animales;méthologies-applications”, 2003, Ed. Barlovatz-Meimom and Adolphe,INSERM editions, Parts. Alternatively, cell extracts are derived from acell bank or a cell culture or harvested as described in the Examplesbelow.

The platelet concentrate and cell compositions of the invention haveproven to be really beneficial in the acceleration and/or promotion ofthe healing process of wounds, even chronic unhealing wounds, leading tosuccessful closures where weeks of conventional therapies had failed andachieving a decrease in infection risks, an improvement in patient'srecovery and comfort, a reduction of medical care costs and a betteraesthetic final result.

The compositions of the invention can of course be also prepared fromplasma derived from several identified donors. The invention is notlimited to autologous biological materials, such as collection ofconcentrated platelets from the wounded's own biological material. Theinvention encompasses the use of biological materials obtained from oneor more third parties, who need not be of the same species as thepatient whose wound is being treated with the wound healant compositiondescribed herein unless bio-incompatibility would result from the use ofsuch third party biological materials.

In one embodiment, the invention provides a process for the preparationof a platelet concentrate composition or a cell composition as describedherein.

In another embodiment, the present invention provides a device for thepreparation of a platelet concentrate composition from whole blood asdescribed herein.

In a further embodiment, is provided by the invention a process for thepreparation of a platelet concentrate composition wherein thecentrifugation step is performed at force between about 1′500 g and upto about 1′700 g for a time selected from about 3 min up to about 15min, preferentially at 1′500 g for about 8 min.

In another further embodiment, is provided by the invention a processfor the preparation of a platelet concentrate composition wherein theseparator tube has an inlet for introducing said whole blood, is held ina vacuum intended to aspirate the whole blood sample, is sterile, has ausable vacuum of 8 to 10 mL and is suitable for undergoingcentrifugation.

In another further embodiment, is provided by the invention a processfor the preparation of a platelet concentrate composition wherein theseparator tube is a polyethylene terephthalate separator tube containinga highly thixotropic gel formed by a polymer mixture and an anhydroussodium citrate at 3.5 mg/mL.

In another embodiment, the present invention provides an isolatedplatelet concentrate composition obtainable from the process accordingto the invention.

In another embodiment, is provided by the invention an isolated plateletconcentrate composition comprising:

-   a) plasma;-   b) platelets at a concentration of at least 300×10⁹ cells/L,    preferably of at least 350×10⁹ cells/L, more preferably of at least    400×10⁹ cells/L;-   c) white blood cells at a concentration of at least 7.0×10⁹ cells/L,    preferably of at least 8.0×10⁹ cells/L;-   d) fibrinogen at a concentration of at least 3 mg/L;-   and wherein the erythrocyte concentration is less than 0.6×10¹²    cells/L, preferably less than 0.5×10¹² cells/L.

In another embodiment, the present invention provides a wound or tissuehealant composition comprising:

-   a) plasma;-   b) platelets at a concentration of at least 300×10⁹ cells/L,    preferably of at least 350×10⁹ cells/L, more preferably of at least    400×10⁹ cells/L;-   c) white blood cells at a concentration of at least 7.0×10⁹ cells/L,    preferably of at least 8.0×10⁹ cells/L;-   d) fibrinogen at a concentration of at least 3 mg/L;-   e) a coagulation activator in a vol. ratio (platelet    concentrate:coagulation activator) of about 10:1 to about 10:3;-   f) optionally an autologous cell extract, such as extract of    keratinocytes, bone marrow cells, fibroblasts, periosteum,    melanocytes and Langerhans cell; fat cells; bone marrow cells;    muscle cells such as myoblasts and satellite cells; osteoblasts;    chondrocytes; periosteal membrane cells; corneal cells; umbilical    cord cells; tendon cells or pancreatic islet cells;-   and wherein the erythrocyte concentration is less than 0.6×10¹²    cells/L, preferably less than 0.5×10¹² cells/L.

In another embodiment, is provided by the invention a process for thepreparation of a wound or tissue healant composition as describedherein.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant composition wherein thecoagulation activator which is admixed is 10% calcium chloride.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant composition wherein thecoagulation activator which is admixed under step b) is a thrombinenriched preparation. A method for preparing thrombin for use in abiological glue is described in U.S. Pat. No. 6,472,162 by the additionof 8 to 20% ETOH to a volume of plasma and this preparation may be usedas a thrombin enriched preparation in the context of the invention.Alternatively, an autologous thrombin serum (ATS) can be used a thrombinenriched preparation in the context of the invention. An autologousthrombin serum according to the invention is obtained by a processcomprising (i) the addition to a patient's whole blood sample (e.g. 8mL) collected in a separator tube of the invention, a 10% of finalvolume of calcium chloride 10% (e.g. 1 mL) and a 10% of the final volumeof a preparation of 95% v. ethanol solution (e.g. 1 mL) and (ii)precipitation for about 30 min at room temperature. After 30 min, acentrifugation at or about 1′500 g for about 8 to 10 min. In a furtherpreferred embodiment, the thrombin enriched preparation and preferablythe autologous thrombin serum is admixed under step b) directly on thewound.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant composition accordingto the invention wherein a further step b′) wherein the activatedplatelet-rich preparation composition (obtained by the admixing of theplatelet concentrate with the said coagulation activator) obtained instep b) may be partially dehydrated by the contact of a wound dressingcovered by a soft hydrophobic layer to avoid contamination withmicro-strings from the dressing in order to obtain a semi-solid gel thatcan be manipulated by appropriate instruments, for example to fill acavity or tissue deficiency, or as a growth matrix (“scaffold”) whilewaiting for the reconstitution of the autogenous extracellular matrix.The obtained wound or tissue healant is particularly useful in a methodfor inducing periodontal regeneration in a wound, a tissue or aperiodontal defect or a cavity.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of keratinocytes.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an autologous extract of keratinocytes.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of skeletal muscle cells such asmuscle progenitor cells or satellite stem cells.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of fibroblasts.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of adipocytes.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of chondrocytes.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of stem cells such as umbilicalcord stem cells.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of tendon cells.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of periosteal membrane orgingival cells.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of corneal cells.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of bone marrow cells.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of osteoblast cells.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of Schwann cells.

In another further embodiment, is provided by the invention a processfor the preparation of a wound or tissue healant or cell compositionwherein the cell extract is an extract of pancreas islet cells.

In another further embodiment, the isolated platelet concentratecomposition, the wound or tissue healant composition, the thrombinenriched serum and/or the cell extract of the invention is/areautologous.

In a further aspect, the present invention provides a kit adapted fortissue regeneration according to the invention wherein the kit furthercomprises separate vials containing ETOH and CaCl₂, syringe holders,clumper and a tip applicator with a dual exit.

In a further aspect, present invention provides a kit adapted for tissueregeneration according to the invention comprising two sterile blisters:

-   (1) one blister comprising accessories for the phlebotomy, separator    tubes of the invention, vials of ETOH and CaCl₂ for the preparation    of an autologous thrombin serum; and-   (2) a second blister comprising accessories for two syringe holders    and clumper, and tip applicator with a dual exit.

In another embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell extract provided under step d) or e) is obtained by a processcomprising the steps of:

-   (A) Providing the said cells in a platelet concentrate according to    the invention;-   (B) Optionally culturing the cells;-   (C) Re-suspending the cultured cells obtained under step (B) into a    platelet concentrate according to the invention.

In a further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell expansion under step (A) is performed in a platelet concentrateaccording to the invention such as the final concentration in plateletscomprised between about 5% and about 40% of the volume of the culturemedium.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) comprises at least one step of plating the cells,for example on a cell culture surface such as a Petri dish or a cultureflask.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention comprisingat least one further step of harvesting the cells after the cell culturestep (B).

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) is performed at 37° C.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) is performed under a gas flow comprising oxygen orair and carbon dioxide, typically the gas flow comprises 95% of oxygenor air and 5% carbon dioxide.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B) is lasting for about 3 up to about 4 weeks.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention whereinduring the cell culture step (B), the cell culture medium is regularlychanged during incubation, typically every about 3 days.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B), comprises at least one exposure step of the cellsto visible light, typically at about 633 nm, during about 10 minutes. Inanother aspect, the exposure step to visible light is repeated once aweek during cell incubation.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell composition is a keratinocyte or fibroblast cell composition andthe cell culture step (B), comprises at least one exposure step of thecells to visible light, typically at about 633 nm, during about 10minutes. In another aspect, the exposure step to visible light isrepeated once a week during cell incubation.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell culture step (B), comprises at least one step of addition ofdiluted platelet concentrate according to the invention such as thefinal concentration in platelets comprised between about 5% and about40% of the volume of the culture medium.

In another further embodiment, the invention provides a process for thepreparation of a cell composition according to the invention wherein thecell composition is a keratinocyte or fibroblast cell composition andthe cell culture step (B), comprises at least one step of addition ofdiluted platelet concentrate according to the invention such as thefinal concentration in platelets comprised between bout 5% and about 40%of the volume of the culture medium.

In another embodiment, the present invention provides an isolated cellcomposition obtainable from a process according to the invention.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a fat cellcomposition such as an adipocyte cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a muscle cellcomposition such as a myoblast cell or a satellite stem cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a corneal cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a cartilage cellcomposition, such as a chondrocyte cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a skin cellcomposition, such as a fibroblast cell or keratinocyte cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a periostealmembrane or gengival cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a tendon cellcomposition, such as tendon cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a stern cellcomposition, such as an umbilical cord stem cell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a bone marrow cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a Schwann cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is a pancreas isletcell composition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein the isolated cell composition is an osteoblast cellcomposition.

In another embodiment, the present invention provides an isolated cellcomposition, wherein cells are at a concentration of about 3×10⁵ toabout 10⁶ cells/ml of plasma or enriched plasma.

In another embodiment, the present invention provides compositions,methods and uses for promoting wound sealing and/or tissue and/or boneregeneration in a wound of a human or a lower animal as describedherein.

In another further embodiment, the present invention providescompositions, methods and uses for promoting wound sealing and/or tissueand/or bone regeneration in a wound of a mammal, preferably human.

In another embodiment, the present invention provides compositions,methods and uses for inducing periodontal regeneration in a wound or aperiodontal defect of a mammal with periodontal disease or othercondition as described herein.

In another further embodiment, the present invention provides a methodfor inducing periodontal regeneration in a wound or a periodontal defector cavity of a mammal with periodontal disease or other conditionwherein the mammal is human.

In another further embodiment, the present invention provides a methodfor inducing periodontal regeneration in a wound or a periodontal defector cavity according to the invention wherein the said therapeuticallyeffective amount of the said wound or tissue healant composition isapplied in a form of semi-solid gel or a growth matrix to the said woundor said periodontal defect or cavity such as described for example inGarg et al., 2000, Dental Implantology Update, 11(6), 41-44.

In another embodiment, the present invention provides a method forpromoting skin tissue regeneration in a scar or wrinkle as describedherein.

In another embodiment, the present invention provides a method forinducing myocardial regeneration according to the invention, wherein thesaid therapeutically effective amount of the said muscle cellcomposition according to the invention is injected into the myocardium,typically into the left ventricule myocardium. Injection can be made asdirect injection or multiple catheter injection. Myoblasts or satellitecells can be engineered ex vivo as described in the present descriptiononto a de-epithelised and UV irradiated human biological amnion andbiocomposite construct, as a monolayer in the present description. Theamnion is then sutured to the ischaemic epicardium in order torepopulate the underlying tissue with stem cells, in order to improvethe contractile power of the ventricular wall and myocytes.

In another embodiment, the present invention provides a method forinducing myocardial regeneration according to the invention, wherein thesaid therapeutically effective amount of the said muscle cellcomposition according to the invention is injected into the myocardium,together with a therapeutically effective amount of fibroblast cellcomposition according to the invention such as the ratiofibroblast/myoblast is of about 30:70.

In another embodiment, the present invention provides a method forinducing myocardial regeneration according to the invention, wherein thesaid therapeutically effective amount of the said muscle cellcomposition according to the invention is applied on the ventricularsurface in the form of an amnion patch preferably incubated into amyoblast and satellite stem cell composition according to the invention.

In another embodiment, the present invention provides a method forinducing corneal regeneration according to the invention, wherein thesaid therapeutically effective amount of the said corneal cellcomposition according to the invention is applied to the corneal tissuein the form of an amnion patch preferably spread on a dissolvablecontact lens.

Said method of treating a wound, a tissue or a disease may include theuse of any of the compositions described herein; it may also include theuse of any composition made by any of the methods described herein.

The methods, the devices and the kit according to the invention presentthe advantages to provide a time-effective and relatively low-cost wayof obtaining platelet concentrates in a single operation that is easy toimplement and adapted to a point-of-care application. The methods of theinvention present the advantage to not only lead to enrichedpreparations wherein the platelets are concentrated in such a high yieldthat is not obtained by known methods but also wherein the content inerythrocytes is much lower than those obtained by known methods for thepreparation of PRP. The compositions of the invention present theadvantage of having a higher content in platelets, a lower content inerythrocytes than PRP obtained by known methods and completelymaintained properties for its subsequent therapeutic use in-vivo. Morespecifically, the ability of the platelets to release the principalgrowth factors involved in tissue regeneration (PDGF, TGF-β, IGF, VEGFand EGF) at levels for several days (or the 7-10 day life span ofthrombocytes) is maintained.

In addition, to the extent the compositions of the invention are madefrom autologous blood, the invention described herein reduces thedisease transmission and immunoreaction risks associated with the use ofthe treatment materials made from biological materials obtained from oneor more third parties.

The invention therefore provides an improved biological wound healingand tissue regenerating material, preferably autologous, promotingtissue such as skin, cartilage and bone regeneration, especiallycicatrisation and/or rejuvenation. The benefits of the inventioncomprise a simple and rapid method of preparation of improved woundhealing and tissue regenerating materials adapted to point-of-careservices and which proved to decrease the healing time, associated painand medical costs. Further, the wound healing and tissue regeneratingmaterial decreases the graft rejection risks and improves graft successrates. Further, the improved wound healing and tissue regeneratingmaterials lead to scars having a much better aesthetic final aspect andto the durable filling of scars and wrinkles.

Typically, cell extracts are obtained from a tissue biopsy wherein thebiopsy is preferably performed the day before the mixture with theplatelet concentrate under step a). The size of the biopsies is adaptedto the aimed therapeutic purpose and the types of cells used in thepreparation of the cell composition according to the invention. Examplesof biopsies are given in the Examples below for different types oftissues.

Examples illustrating the invention will be described hereinafter in amore detailed manner and by reference to the embodiments represented inthe Figures.

EXAMPLES

The following abbreviations refer respectively to the definitions below:

-   ATS (autologous thrombin serum); BU (Baxothrobin unit); DMEM    (Dulbecco's minimum essential medium); DMSO (Dimethyl Sulfoxide); EC    (Enriched clot); FCS (fetal calf serum); HT (healing time); IU    (International Unit); PBS (Phosphate Buffered Saline); PET    (polyethylene terephthalate); PRP (platelet-rich plasma); PPP    (platelet-poor plasma); USP (United States Pharmacopoeia); cm    (centimeter); dL (decilitre); g (gram); Gy (gray); J (Joule); L    (liter); min (minute); mm (millimetre); M (molar); mL (millilitre);    nm (nanometre); rpm (Rotation per minute); Vol. (volume).

General Procedures & Conditions

To determine the effectiveness of compositions of the invention inpromoting wound healing and/or bone and/or tissue regeneration, thefollowing experiments are performed.

Whole human blood samples are collected in a separator tube according tothe invention. A separator tube according to the invention is forexample an approximately 15 mL glass tube (16 mm diameter and 130 mm inlength) containing 3 mL of polyester-based thixotropic gel as well as 1mL of sodium citrate solution at 0.1 M and containing a usable vacuum ofor about 8.5 mL. This separator tube constitutes a ready-to-use devicefor the preparation of a platelet concentrate composition of theinvention (also called RegenTHT™ (Thrombocyte Harvesting Tube) fromRegen Lab, Switzerland).

Another example of a separator tube according to the invention is a tubeof approximately 10 mL in PET (polyethylene terephthalate) containing 1mL of a thixotropic gel comprising a polymer mixture and anhydroussodium citrate deposited on the inner surface of the tube by spraying(about 3.5 mg per mL of blood) and containing an usable vacuum of orabout 8 mL, constitutes a ready-to-use device for the preparation of aplatelet concentrate according to the invention (also called RegenBCT™(Blood Cell Therapy) from Regen Lab, Switzerland).

These tubes are sterilized by irradiation (such as prescribed by ISO11137, UNI EN ISO 11737-2, UNI EN 552, UNI EN 556) and hermeticallysealed by a traditional cap such mottled bromobutyl conventional rubberstopper for the glass tube and a chlorobutyl stopper having apolyethylene cover for the operator safely.

Then, the separator tube is centrifuged at or about 1′500 g up to orabout 2′000 g, for about 3 to 10 min, i.e. of or about 2′500 rpm up toor about 3′800 rpm with a centrifuge with a swinging rotor, having aradius of 20 cm, in case of a centrifuge having a rotor with a fixedangle of about 45°, the centrifugation time should last for at leastabout 15 min.

After centrifugation, the platelet concentrate is collected for use intherapeutic or cosmetic applications of the invention or for thepreparation of further compositions containing the obtained plateletconcentrate through the mixture with further agents such as cellextracts, preferably autologous (e.g. keratinocytes, fibroblasts, bonemarrow cells, osteoblasts, chondrocytes, myoblasts, conical cells,schwann cell, fat cells, umbilical cord stem cells, tendon cells,pancreas islet cells, ligament and gingival cells, periosteal membranecells) and/or bone substitutes and/or coagulation activators.

For the therapeutic applications, a kit according to the inventionadapted for tissue regeneration is used wherein the kit (also calledRegenKit™) comprises two sterile blisters comprising:

-   -   one blister (RegenPRP™) comprising accessories for the        phlebotomy, separator tubes of the invention (RegenTHT™ or        RegenBCT™), optionally vials of ETOH and CaCl₂ for the        preparation of an autologous thrombin serum (RegenATS™).    -   one blister (RegenApplicator™) comprising two syringes (e.g. 1        mL and 1 or 3 mL), holders and damper and a tip applicator with        a dual exit.

For the preparation of cell compositions, according to the invention,the cells are prepared according to the general protocol as follows:

a) Biopsy

A biopsy of the corresponding tissue is obtained under sterileconditions using standards methods adapted to the specific cell thatwill be collected. The cells are used extemporaneously or optionallyafter ex-vivo culture and cell proliferation as follow.

b) Ex-Vivo Culture and Cell Proliferation

Cells used for the preparation of cell compositions according to theinvention, such as keratinocytes, bone marrow cells, fibroblasts;periosteum or corneal cells, such as corneal limbal stem cells;melanocytes and Langerhans cell; fat cells; muscle cells such asmyoblasts and satellite cells; osteoblasts; chondrocytes; umbilical cordcells; Schwann cells, tendon or pancreatic cells are expanded in a cellcarrier medium (e.g. DMEM or Ham's) on plates (e.g. Petri dishes orculture flask) coated with a platelet concentrate according to theinvention, preferably autologous, enriched with fibronectin. The culturemedia may be enriched preferably with DMEM for example in the case ofkeratinocytes. For cells such as bone osteoblasts, chondrocytes andmyoblasts, enzymatic digestion of the corresponding tissue in presenceof for example collagenase or trypsin is necessary before plating.Incubation on the plates is performed at 37° C. under a gas flow of 95%oxygen or air and 5% carbon dioxide. Typically, incubation time varyfrom 10 to 20 min. The cell expansion may be increased (like for examplein the case of myoblast, fibroblast and chondrocyte cells) byphototherapy (e.g. light exposure at 633 nm of about 10 min at 2 J/cm²,once a week during the incubation phase).

The explants may be cultured in Petri dishes or culture flask usingair-lifting technique (Molnar et al., 1996, Tissue & Cell, 28:547-556)and air interface method (Meller et al., 2002, Br. J Opht., 86, 463-471)with half of the explant exposed to air. The culture medium is changedregularly during incubation, such as every 3 days. The expansion of thecells in a 2D mode as planar monolayers, is obtained for example formyoblast, fibroblast and chondrocyte cells. A 3D cell growing patterncan be obtained for example for corneal, myoblast, fibroblast,chondrocyte, adipocyte and keratinocyte cells, by adding dilutedautologous platelet concentrate composition according to the inventionat about 5 to about 40% volume of plasma or enriched plasma to theculture medium. Typically, the addition of diluted autologous plateletconcentrate composition according to the invention is performed 2 orthree times during the incubation time. The 3D biological scaffold thenobtained allows to enhance the extra-cellular matrix which is useful forautologous stern cell transfer.

After incubation, cells are then released from dishes with gentletrypsin digestion that lifts off the cells and allows them to bepelleted.

c) Cell Quality and Safety Check

The cell viability in the so-obtained cell preparation is checked bymicroscopic cell count, flow-cytometer cell count together withimmunochemistry on tissue markers by standard techniques. Cell viabilityis also tested via trypan-blue just after cell release by trypsin.Safety of the preparation is also checked through contamination checkvia microbiology assay to exclude contamination with viruses or bacteriaand to avoid transfer of zoonotic infections. The use of FCS is avoidedthus preventing transmission of Mad Cow Disease.

d) Administration of the Cell Preparation

The cell preparation obtained above is placed in autologous plateletconcentrate composition according to the invention as cell carriervehicle for transport before delivery to the patient. Then, the cellpreparation obtained above is injected or transplanted into the patient.The injection or transplantation mode has to be adapted to the type ofcells contained in the cell preparation according to the invention andto the aimed therapeutic or aesthetic effect. More details are given inthe Examples below on the method for the preparation and use of the cellcompositions according to the invention more specifically, depending onthe type of cells and aimed therapeutic or aesthetic effect.

Keratinocyte cell or fibroblast cell preparations according to theinvention may be used readily after collection or after cell culture asdescribed above. However, the cell preparations according to theinvention are preferably prepared after cell culture as described above.

The cell preparations according to the invention present a betterviability and stability (including integrity of cell propertiespreserved such as ability the synthesize proteins and deliver growthsfactors) than cells prepared in a medium without autologous plateletconcentrate composition according to the invention. Further, cellproliferation so obtained is enhanced: cells grow faster (about 3 to 5days quicker) and are denser compared to control mediums and serumstarved media. The advantage of the process for the preparation of acell composition according to the invention is that the same autologousmedium is used as vector for cell culture, cell preservation, cellinjection, vector for cell bio-stimulation and tissue regeneration.

Example 1 Preparation of an Autologous Platelet Concentrate

Separator tubes of the invention are beforehand tested for the goodtolerability, the non-toxicity and the non-mutagenicity of thethixotropic gel according to norms ISO 10993-11, ISO 10993-10, ISO10993-12 and ISO 10993-3.

About 8.5 to about 10-mL of human blood sample are collected within theseparator tube of the invention, where the blood is aspirated by thevacuum. The mixture is then centrifuged at approximately 3′800 rpm forabout 3.5 min. The platelet-rich plasma is then collected.

The analysis of the platelet concentrate obtained by the method of theinvention has shown that it contains 2 to 4 times the normal levels ofplatelets and growth factors, compared to a natural blood clot, whilemaintaining normal levels of fibrin and fibrinogen and containingpractically no blood cells (<1% hematocrit, compared to 35-50% in anormal blood clot and 15-20% in platelet-rich plasma obtained from knownmethods of preparation). The study shows also the presence of leukocytesglycoprotein fibronectin and this demonstrates that the coagulatingproperties are preserved.

The composition of the platelet concentrate (also called RegenPRP™ fromRegen Lab, Switzerland) compared to whole blood, whole plasma andplatelet-poor plasma is presented in table 1 below:

TABLE 1 White Red Hemo- Blood Cells Blood Cells globin Platelets Sample(×10⁹/L) (×10¹²/L) (g/dL) (×10⁹/L) Complete blood 6.6 4.43 13.5 269Complete plasma 2.8 0.04 0.4 305 Platelet-rich plasma 14.2 0.57 2.1 570Platelet-poor plasma 0 0 0.1 116

The experiment was repeated on several patients and it was observed thatthe obtained platelet concentrates present reproducible concentrationsfor platelets (at least 300×10⁹ cells/L), white blood cells (at least7.0×10⁹ cells/L), fibrinogen (at least 3 mg/L) and erythrocytes (lessthan 0.6×10¹² cells/L).

The platelet yield obtained by such the method of the invention has beenmeasured (90-99%) and shows to be drastically increased in comparisonwith the platelet yields (30-62%) obtained from known methods ofpreparation described in Marx et al., 2004, above.

In addition, it has been shown through ELISA kits (R&D Systems, Inc.)and the response to coagulation activation of the platelet concentrateof the invention, that the activity of coagulation factors is preserved:the concentration of D-dimers (fibrin breakdown products), known markersof coagulation activation, and the lysis process are stable andtherefore the coagulation properties of the platelet concentrate are notweakened by the process of the invention.

The levels of growth factors (PDGF, EGF, TGF-β and VEGF) from theplatelet concentrate of the invention are demonstrably stable for aperiod of at least 72 hours (4 days) when stored at room temperature inthe sterile separator tube of the invention. The evolution of growthfactors PDGF BB, EGF and VEGF over 72 hours is presented on FIG. 1.

The properties of the platelet concentrate according to the inventionmake it possible to envisage preparing platelet concentrate obtainedusing the invention's procedure, one to several days before a reparativesurgery, in order to reduce the workload in the operating room andspeed-up the surgical procedure.

For subsequent therapeutic use, the autologous platelet concentrate isgenerally mixed with a conventional coagulation activator such as athrombin activator (e.g. calcium chloride pour example at 10%),optionally mixed with a fibrinogen activator such as thrombin,preferably homologous (e.g. 10 UI to 100 IU per mL of plasma),batroxobin (e.g. 20 BU per mL of plasma) or a thrombin enrichedpreparation.

Example 2 Therapeutic Use of the Autologous Platelet Concentrate of theInvention a) Patients:

Three patients presenting chronic unhealing wounds are selected:

-   -   One 88-year-old patient (Patient 1) suffering from multiple        locations Kaposi's angiosarcoma on lower limbs and from a        radio-induced necrosis on the left leg. The radio-induced        necrosis was resulting from radiotherapy treatment. After 12        months after the end of the low-voltage X-ray treatment, the        necrosis was consisting in a deep superinfected ulcer surrounded        by a scab (35×25 mm). The wound had been previously        unsuccessfully treated with various treatments such as with        steroids and healing creams.    -   One 81-year-old patient (Patient 2) suffering from a vertex        spinocellular carcinoma was presenting a cutaneous ulceration        (about 10 mm diameter) with peripheral dyskeratosis without any        infection sign resulting from a biopsy-resection and a        post-surgical radiotherapy (total close of 52 Gy).    -   One 60-year-old patient (Patient 3) having received a        pre-surgical irradiation (7 Gy) for tibia and fibula synostosis        on the right leg was presenting a radio-induced necrosis        consisting in a deep ulcer (50×30 mm diameter) without        inflammation.

b) Treatment:

8.5 mL of blood sample is taken from each patient and centrifuged in aseparator tube as described in Example 1, according to the protocol asdescribed in Example 2. The resulting platelet concentrates are thenmixed with calcium chloride at 10% vol. Each autologous plateletconcentrate composition is then applied on the radio epidermitis woundsite of the corresponding patient. The wound is then covered andprotected with humid compresses (Day 1).

Between days 3 and 5, the wound status is checked and the wound dressingis changed. At day 7±1, a new application of a new autologous plateletconcentrate preparation of the invention is performed. If needed, thesame treatment sequence is followed with the same time intervals tillthe complete cicatrisation of the wound.

c) Healing Effects:

Patient 1: Slow and regular healing of the ulcer. Complete cicatrisationafter 189 days.

Patient 2: Very quick healing obtained in 21 days.

Patient 3: Progressive and regular healing. Complete cicatrisation after41 days.

These results show the benefit effect of the platelet concentratecomposition of the invention in the healing of chronic radio-inducedulcers, even in the case of those which were resistant to previous topictreatments and in the absence of any allergic reaction.

Example 3 Therapeutic Use of the Autologous Platelet Concentrate of theInvention in Combination with an Autologous Thrombin Enriched Serum

To activate coagulation, an alternative to the mixture of the plateletconcentrate of the invention with a thrombin activator before the use ona patient, as described in Example 1, is the combination of the plateletconcentrate of the invention with a fibrinogen activator such as athrombin enriched composition and preferably with a thrombin serum (e.g.autologous) according to the invention.

a) Preparation of an Autologous Thrombin Serum (ATS)

An autologous thrombin serum to be used as a thrombin enrichedpreparation in the context of the invention is prepared by a processwhich comprises the addition to a patient's whole blood sample (e.g. 8mL) collected in a separator tube of the invention as described inExample 1, a 95% v. ethanol solution (e.g. 1 mL) and calcium chloride10% (e.g. 1 mL). The mixture (also called RegenATS™ from Regen Lab,Switzerland) is then allowed to precipitate for about 30 min at roomtemperature.

After 30 min, almost 80% of the anti-thrombin (among other proteins likefibrinogen) is precipitated; then the tube is centrifuged at or about1′500 g for about 8 to 10 min and the autologous thrombin serum is readyfor use in combination with the platelet-rich concentrate of theinvention.

b) Combined Preparations

One of the originality of this process is that after the initial step ofincubation of the autologous thrombin serum preparation process (e.g. atleast about 30 min), the separator tubes of the invention containingrespectively the autologous thrombin serum preparation and the plateletconcentrate preparation can be centrifuged simultaneously in order toget the two blood extract preparations ready for use at the same time.

c) Combined Use

To allow the polymerization of fibrinogen into a fibrin mesh (whichoccurs during the coagulation process) to occur only at the moment ofapplication of the platelet-rich preparation on the wound, the plateletconcentrate composition and autologous thrombin serum (coagulationactivator) are applied simultaneously at a vol. ratio of about 10:1 toabout 10:3 (concentrate to coagulation activator ratio) to the wound.

The simultaneous delivery of both preparations is achieved for exampleby a device comprising two syringes (e.g. 10-mL syringe for the plateletconcentrate composition and a 1-mL or 3 mL syringe for the thrombinserum), that releases the preparations simultaneously so that they mixand polymerize upon contact with the wound.

Example 4 Therapeutic Use of the Autologous Platelet Concentrate of theInvention in Combination with Skin Cell Extract

A total of 35 patients having received a skin graft (representing lessthan 15% of the skin surface) have been included in the study. Patientstreated with immunosuppressants or corticoids or with renalinsufficiency or severe peripheral artheropathy were excluded.

All the following manipulations are performed under the strict rules ofasepsy and sterility.

Group A: 13 Patients a) Preparation of Platelet Concentrate

A 8.5 mL sample of whole blood from each patient (from a higher limbwhere no perfusion is present) is collected in a separator tubeaccording to the invention. The separator tube with the whole blood isimmediately centrifuged during about 8 min at 2′800 rpm. Before theenriched plasma (PRP) is collected, the operator discards the half or 2mL of the supernatant and then re-suspends the platelets in theremaining plasma. The platelet-rich concentrate is then transferred to asterile tube maintained at a temperature of 37° C.

b) Wound Coating

The autologous platelet concentrate of the invention (also calledRegenPRP™) is mixed with a solution of calcium chloride 10% in a ratio10:1 and the graft donor site (where skin was removed) of eachcorresponding patient is coated with the autologous correspondingmixture in order to obtain coagulation of the platelet concentrate onthe wound.

Group B: 8 Patients a) Skin Cell Sampling on the Patient

Keratinocytes are extracted from each of the patients from this group. Athin healthy skin sample (about 2 cm²) is removed from each patient andwashed three times in a PBS solution. The washed biopsy is thendeposited in a Petri dish containing trypsin and cut into very smallfragments (0.5 cm*0.5 cm) with a scalpel. The skin fragments are thenincubated during 45 min at 37° C. on a stirring device in 20% volume ofautologous platelet concentrate composition according to the invention,also called RegenPRP™^(,) obtained above. The supernatant is thencollected, centrifuged and cells are re-suspended in a PBS solution. Thekeratinocytes count is determined under microscope. Finally, theobtained keratinocytes were re-suspended in the autologous plateletconcentrate according to the invention (5-40% vol.) from thecorresponding patient.

b) Preparation of Platelet Concentrate

The procedure is the same as for Group A:

c) Wound Coating

The keratinocyte suspension (also called RegenExtracell™) is applied assoon as ready (the entire preparation not exceeding a day) on the woundon the same way as described in the case of Group A.

Control Group: 14 Patients

The graft donor site of each patient of this group is coated with anon-therapeutic compress (Jelonet®).

Randomization and Treatment

In the surgery bloc, after the graft skin removal, the donor site iscoated with a temporary compress soaked with an adrenaline solution (1ampoule of 1 mg/mL of adrenaline diluted in 500 mL NaCl 0.9%) anddepending on the randomization table, the donor site is treatedaccording to the three following methods:

Groups 1 and 2: Coating of the wound with the respective wound healingcomposition and covering of the wound with a non-therapeutic compress(Jelonet®).

Group 3: Direct covering of the wound with a non-therapeutic compress(Jelonet®).

The compresses are then covered with Kerlix® bands and elastic bandssuch as “Velpeau”.

Treatment Efficacy Criteria

The efficacy of the treatment is evaluated according to 3 criteria:

-   -   The time needed for the complete cicatrisation of the treated        site (healing time or HT in days)    -   The epitheliazation (evolution of the cicatrisation progress)        measured at day 5 after the treatment according to 7 degrees:    -   0: Absent    -   1: Slight    -   2: Moderate    -   3: Important    -   4-7: Very important, increasing degrees of importance;    -   The pain evaluated at day 5 after the treatment by the patient        him/herself, generally at the time of compress change on a scale        from 0 to 10 (0: no pain and 10: extreme pain).

The compress is opened at day 5 post-surgery to allow the evaluation ofthe quality of the treatment and covered with new Jelonet® compressescovered with dry compresses.

The compress is then changed very two days till the completecicatrisation. Any side effects or medical complications are watchedduring the whole duration of the cicatrisation process.

Results

The results of the treatments for each patient group (Control group: C,Group A: RegenPRP™, Group B: RegenExtracell™) are presented on FIG. 2 interms of healing time in days (HT), pain at day 5 (P) andepitheliazation at day 5 (E). The dotted line indicates when the firstbandage is changed at day 5.

The cicatrisation process is clearly stimulated by the use of theplatelet concentrate of the invention as compared to the control group.The quality of the cicatrisation is also better in the case of the useof platelet concentrate of the invention. In addition, the pain at thedonor site is dramatically reduced in the case where the plateletconcentrate of the invention was used as compared to the control group.

All the beneficial effects of the platelet concentrate of the inventionare increased when a mixture of keratinocytes suspended in the plateletconcentrate of the invention is used.

The mean healing time is of 7 days for the group treated with a plateletconcentrate of the invention and 5 days when keratinocytes are suspendedin the platelet concentrate as compared to an average of 12 days in thecontrol group.

Tolerability was excellent and no side effect or allergy has beendetected.

This shows that the platelet concentrate of the invention alone orcombined with keratinocytes is very efficient in accelerating the woundhealing process and not only decreases the pain, but also theinflammatory reaction and improves the final aspect of the scar.

Alternatively, using the same process of dissociation, skin cells can beplaced in a Petri dish coated with the autologous platelet concentratecomposition according to the invention, also called RegenPRP™^(,)obtained above and cultured for 2 to 5 days. Then, before the graft, theobtained skin cell preparation may be sprayed onto the wound, in orderto prepare the site for a better bio integration of the implanted cells,and a better expansion in vivo.

Example 5 Cosmetic Use of the Autologous Platelet Concentrate of theInvention

An autologous platelet concentrate composition is prepared as describedin Example 1.5 mL of this platelet concentrate composition (also calledRegenACR™: (Autologous Cell Rejuvenation) from RegenLab, Switzerland) isinjected subcutaneously in a wrinkle groove as wrinkle filling material,in the same way as commonly done with other wrinkle filler such ashyaluronic acid. The deepness of the wrinkle is progressively decreasingwithin the first weeks after the treatment and at the site of injection,a very clear regeneration of the area is obtained with an optimal resultat two to three months. As opposed to what observed with other wrinklefilling materials, neither inflammation, nor swelling is observed at thesite of injection and the benefit is durable as opposed to hyaluronicacid which is bio-resorbed after 4 to 6 months.

Known methods to study the effect autologous platelet concentratecompositions of the invention on wrinkle deepness can be used to such asa three-dimensional reconstitution of skin relief by opticalprofilometry (stylus method) (Grove et al., 1989, J. Am. Acad.Dermatol., 21:631-7) or by laser microscopy on silicon skin replicas.Another method consists in the in vivo quantification of the skinsurface “Surface evaluation of living skin” or “SELS” through theanalysis of images in UV light (Tronnier et 1997, Akt. Dermatol.,23:290-295). Another method for the surface evaluation of living skin isbased on an optical system with a CCD camera measuring the four skinparameters: roughness, scaling, smoothing and wrinkling (Fluhr et al.,1995, Akt. Dermatol., 21:151-156). Dep dermal augmentation can beassessed by ultrasound, Dermascan®, Denmark).

Other examples of cosmetic use of the autologous platelet concentrate ofthe present invention include:

Admixing the platelet concentrate according to the invention with acream, preferably an emulsion, before application to a wound, aftersurgery or on healthy skin. During the absorption process, the plateletpreparation is carried into the skin by the cream or emulsion in orderto amplify the hydrating benefit and to bio-stimulate the regenerationor rejuvenation of the skin.

Using a hydrogel like the Albugel (EP 1 543 846) preparation of 100%Albumin or any other hydrogel resulting from the reticulation of Albuminand other chemical compound like polyethylene glycol or any otheringredient, using a paper based highly hydrophilic, a carrier to leavein contact with the skin until the platelet rich plasma is absorbed.

Example 6 Autologous Muscle Cell Association Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention whereinskeletal muscle cells (muscle progenitor cells or satellite stem cells)are provided under step (d) or (e).

a) Myoblast Progenitor Stem Cells

Skeletal muscle biopsy is obtained from the Vastus lateralis andmeasures 7×3 cm. Muscle is primed the day before biopsy, withintra-muscular injection at proposed biopsy site (10 by 15 cm skin areaon lateral aspect of thigh overlying the vastus lateralis muscle andjust above the knee joint, on either side) with Decadon and Marcaine(long acting Lignocaine). Muscle is diced and enzymatically digestedwith combination of collagenase, pronase and trypsin (Worthington).Enzyme action is neutralised using patients serum in DMEM culturemedium. Muscle explants are plated Petri dishes coated with theautologous platelet concentrate composition according to the invention,also called RegenPRP™ (prepared as described in Example 4) and incubatedin 95% oxygen and 5% carbon dioxide at 37° C. for 3 to 4 weeks. Desminor CD-56 expression is used as myoblast marker to identify myoblastsfrom fibroblasts. Myoblast progenitor cell proliferation in 3D is shownon FIG. 3.

Cell proliferation can be enhanced by photo-light exposure at 633 nm of2 J/square centimetre for 10 min during culture. The day oftransplantation (e.g. after 3 to 4 weeks of incubation), the skeletalmuscle cells are released are released by trypsin and placed in theautologous platelet concentrate composition according to the invention,also called RegenPRP™ (prepared as described in Example 4). Injectionsinto the myocardium can be made as direct injection or multiple catheterinjections into the left ventricle myocardium. The myoblast cellpreparation according to the invention is useful for cardiac disorderssuch as heart regeneration, treatment of heart failure, chronic cardiacfailure, ischemic and non-ischemic cardiac failure and non-ischemiccardiomyopathy. Ejection fraction can be improved by 9% for cardiacrecipients of skeletal myoblasts.

The above cell preparation may also be useful for in the treatment ofurinary incontinence (myoblast cell extracts prepared as described aboveand injected into the bladder neck), reflux oesophagitis orgastro-oesophageal reflux disorder (myoblast cell extracts prepared asdescribed above injected into the lower oesophageal sphincter) and analincontinence (myoblast cell extracts prepared as described above andinjected in para-anal area).

Alternatively, a combined preparation of fibroblast and myoblast may becarried out (fibroblasts are present in the muscle biopsy and sproutfrom the perimysium alongside the myotubes and satellite stem cells).

In case of the treatment of cardiac disorders, a mix of fibroblast cellpreparation and myoblast cell preparation (obtained as indicated above)is inserted into the myocardium in a ratio fibroblast/myoblast of about30:70.

For bladder neck incontinence treatment, a separate culture offibroblasts is made at the same time as the myoblasts as described aboveand the fibroblast cell preparation is injected para-urethrally andmyoblast cell preparation is injected into the rhabdosphincter, underultrasound control.

b) Satellite Stem Cells

Myoblasts and satellite stem cells are cultured ex vivi in presence ofautologous platelet concentrate composition according to the invention,also called RegenPRP™. Cell proliferation priming is observed after7-days of primary culture.

Cells are then harvested after incubation of about: 3-4 weeks and placedin tissue culture medium (DMEM plus 5-10% vol. autologous plateletconcentrate composition according to the invention) containing a humande-epithelialized amnion patch measuring 4×4 cm and the autologousplatelet concentrate composition according to the invention, also calledRegenPRP™ (prepared as described in Example 4). The preparation is thensubjected to UV irradiation for 10 min. During incubation (typicallyabout 2 to about 3 weeks), the cells spread over the amnion constructand form a monolayer. Viability and monolayer progress is assessed bytwice weekly biopsy of patch edge and histological assessment forthickness of monolayer.

The day of transplantation (e.g. after about 3 to 4 weeks ofincubation), the ventricular surface is spread with the autologousplatelet concentrate composition according to the invention, also calledRegenPRP™ (prepared as described in Example 4) and then the patchobtained above is placed with cells down side onto a raw surface of theischemic ventricle in order to allow the stem cells on the patch topopulate the ischemic segment after ventricular injection. Cellretention is maintained by the amnion that is inert and induces noimmunological reaction.

The satellite stem cell preparation according to the invention is usefulfor heart regeneration and treatment of heart failure as tissueengineering preparation for cardio myoplasty.

Example 7 Autologous Fibroblast Cell Association Preparation

Example of autologous fibroblast cell association according to theinvention can be prepared by using the process according to theinvention wherein dermal fibroblast cells are provided under step (d) or(e).

Dermal fibroblasts are isolated and expanded according to the followingprocedure:

One month before biopsy, the prime donor skin area (behind an ear ofanterior axillary fold, e.g. non solar aged area) is treated withvitamin A cream to activate the dermal fibroblasts. A skin biopsy of10×6 mm full thickness is performed and dissected under microscope toremove all epithelium. The-epithelialize biopsy (dermis) is then cutinto 3×3 mm blocks as explants. The papillary dermis is then placedupwards and cultured using air-lifting technique (Molnar et al., 1996,above) and air interface (Meller et al., 2002, above) with half of theexplant exposed to air. The explants are plated (e.g. 6 explants perwell) in DMEM and cultured at 37° C. at 95% oxygen and 5% CO₂ for about3-5 up to about 9 days in Petri dishes or culture flask. The medium ischanged every 3 day. The fibroblasts expansion in 2D mode as planarmonolayers, as static growth is observed during incubation. At days 7 to9 after the start of incubation, a change in proliferation and phenotypepattern to 3D is obtained by adding diluted 5-10% autologous plateletconcentrate composition according to the invention, also calledRegenPRP™ (prepared as described in Example 4) to the culture medium:cells are primed with RegenPRP™ (0.2 ml per well) just to cover base.Cells grow then as a 3D fibrin gel matrix (FIG. 3). Cells thendifferentiate to form biological scaffold or network in fibrin gel suchas shown on FIGS. 4a and 4 b. Cell number is measured by daily countingunder a grid and to assess apoptosis: use inverted microscope(Olympus®).

After 3 to 6 weeks of incubation, the cells are harvested from thefibrin gel. Cell viability is assayed with classical Trypan blue methodand with bacteriological evaluation, including virus contamination.

The expanded fibroblast cell extract obtained above is placed in asyringe in presence of autologous platelet concentrate compositionaccording to the invention, also called RegenPRP™ and the preparation isinjected into face wrinkles, more specifically under the wrinkles.Injections must be performed over the whole face to cover forehead,jowls, molar region, cheeks, chin and neck.

Cell expansion may be increased by photo light exposure of cell cultureat 633 nm. The fibroblast cell preparation according to the invention isuseful for facial rejuvenation, amelioration of facial wrinkles andrhytids, treatment of skins damaged by radiations (radiodermatitis orsun damaged skin), aged skins or burned skins and/or in the ameliorationof facial wrinkles, rhytids, acne (especially after dermabrasiontreatment), burns, rubella or small pox scars, vitiligo, lipoatrophy orlypodystrophy, such as AIDS-related lypodystrophy; Kaposi's sarcoma,skin keloids or Dupuytren's palmar fibromatosis and/or in skinrejuvenation treatments.

Example 8 Autologous Fat Cell Association Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention wherein adiposestern cells are provided under step (d) or (e).

Adult adipose stem cells are isolated by standard culture method in5-10% vol. an autologous platelet concentrate composition according tothe invention, also called RegenPRP™. The preparation is then injectedwith an applicator into patients suffering from tissue deficiencies,such as post traumatic deficiencies or aged-related deficiencies forpatients being around about 40 years-old.

The fat cell preparation according to the invention is useful for thetreatment of lipoatrophy such as in HIV/AIDS patients and otherscongenital hemiatrophy of the face.

Example 9 Autologous Chondrocyte Cell Association Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention whereinchondrocyte cells are provided under step (d) or (e).

Cartilage is isolated from the donor's knee (biopsy size 10×5 mm) anddiced. The cartilage chondrocyte cells are cultured for 4-6 weeks inmedium enriched with an autologous platelet concentrate compositionaccording to the invention, also called RegenPRP™^(,) Cartilage cellsare then released by enzymatic digestion (collagenase and pronase). Thecell preparation is then incorporated surgically into the patient withdeep chondral defects and damage.

The chondrocyte cell preparation according to the invention is usefulfor the treatment of deep cartilage damage and erosion or arthroscopy.

Another example of the use of a chondrocyte cell preparation accordingto the invention is the use in rhinoplasty without surgery by a singleinjection procedure: A patient suffering from congenital cartilage noseatrophy.

The day before injection, a biopsy of the cartilage of the ear 0.4*0.4cm is performed and placed in a sterile recipient filled with DMEM andantibiotic. The biopsy is treated with enzymatic digestion includingtrypsin and collagenase. The released chondrocytes are then re-suspendedin the autologous platelet concentrate composition according to theinvention where 10% CaCl₂ have been added.

The patient receives first a local anesthesia, and nose disinfection.Then, the above chondrocyte preparation is injected on the cartilagesurface and or periosteum membrane of the site requiring augmentation ofvolume or lift. In a second phase, autologous platelet concentratecomposition according to the invention where 10% CaCl₂ have been addedis injected into the superficial part of the nose skin, in order tobiostimulate regeneration and the rejuvenation of the skin. After onehour, the injection is achieved and the patient could return home. Anexceptional recovery of viable cells is observed: the amount ofchondrocyte cells and plasma cells recovered and injected was about 10⁹cells.

The chondrocyte cell preparation according to the invention is thereforeuseful for the treatment of nasal cartilage defects, without surgicalprocedure, but only by injection.

Example 10 Autologous Umbilical Cord Stem Cell Association Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention whereinumbilical cord stem cells are provided under step (d) or (e). Umbilicalcord stem cells are isolated and then cryo-preserved and used to treatblood disorders.

The umbilical cord stern cell preparation according to the invention isuseful for the treatment of haematological diseases (like Thalassaemia).

Example 11 Autologous Tendon Cell Association Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention wherein tendoncells are provided under step (d) or (e).

Tendon fibroblast cells are isolated according to procedure standardprocedures in 5-10% vol. of autologous platelet concentrate compositionaccording, to the invention. The tendon fibroblast cells are culturedfor about 1 to about 3 weeks in culture medium enriched with anautologous platelet concentrate composition according to the invention,also called RegenPRP™. The cell preparation is then injected into thepatient at the injury site (e.g. tendon torn, arthritic area). Theinjection can be guided by echography, for localisation of the damagedsite, and better graft of the implanted solution.

The injection of the tendon fibroblast cell preparation may also beperformed next to rotator cuff in shoulder: first the rotator cuff tearis repaired arthroscopically, then the tendon fibroblast cellpreparation is injected via a long catheter onto the sutured area. Thisimproves the healing of the tendon fibroblast at the edge of the rotatorcuff, prevents haematoma in confined space under the acromion, preventsfrozen shoulder by speeding up healing and enhancing rehabilitation and,joint movement.

The tendon cell preparation according to the invention is useful for thetreatment of tendons torn, arthritis in joint caused by traumas or byaging, rotator cuff in shoulder.

Example 12 Autologous Ligament and Gingival Cell Association Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention whereinperiosteal membrane and gingival cells are provided under step (d) or(e).

Under general and local anesthesia, periosteum (approximately 10×10 mm)is aseptically harvested from the buccal side of the mandibular body infour healthy female beagle dogs. The harvested periosteum is cut into3×3 mm pieces. The tissues are placed directly on a 6-well plate andcultured (for about 3 to about 6 weeks) in a humidified atmosphere of 5%CO₂ and 95% air at 37° C. in a culture medium enriched with anautologous platelet concentrate composition according to the invention,also called RegenPRP™. The periosteal membrane and gingival cells areisolated by enzymatic digestion and cultured by static technique.

Typically, 6 weeks of culture was sufficient to obtain appropriateperiosteal membrane thickness for grafting.

Patients are separated into two groups: (1) a control group whichreceives an autologous platelet concentrate composition according to theinvention without periosteal membrane cells and (2) a treatment groupwhich receives the periosteum membrane cell preparation obtained above.The autologous platelet concentrate composition (in control group) andthe cell preparation (for the treated group) are respectively injectedinto the patient at the injury site.

12 weeks after operation, the periosteal membrane cell preparation hadcompletely disappeared in both control and treatment group. Boneregeneration in the treatment group with cultured periosteum membranewas significantly greater than that in the control group: the thread ofdental implants was almost covered with the regenerated bone, while mostof the thread in the control group was exposed. The treatment groupdemonstrated relatively thick and dense lamellar bone formation frombottom to top of the created defect. Thick layers of woven bone wereattached to the implant surface and osteoblast like cells were observedaround the surface. Although abundant neovascularization was observed inthe bone matrix, inflammatory cells were rarely observed. The borderbetween the regenerated and original bone was not clear. In the controlgroup, specimen exhibited thin cortical bone formation at the dehiscencedefect. Scarce woven bone was observed between the implant surface andthe cortical bone, and few osteocytes and osteoclasts were observedwithin the bone matrix and at the surface, respectively. Bone densitywas significantly higher in the treatment group than the control group.The mean LF values were 77.58±1.14% and 37.03±4.63% in the treatment andcontrol groups, respectively (P<0.05).

The periosteal membrane and gingival cell preparation according to theinvention is useful for the treatment of periodontal disease and drysockets, especially in promoting bone regeneration at sites of implantdehiscence.

Example 13 Autologous Corneal Cell Association Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention wherein cornealcells are provided under step (d) or (e).

A biopsy is taken from the epicanthis on the edge of the cornea and thecorneal limbal stern cells were expanded for autologous transplantationin the same person after 4 weeks of culture in Petri-dishes or flaskscoated with an autologous platelet concentrate composition according tothe invention, also called RegenPRP™.

The corneal corneal cultured stem cells (of limbal origin) may be exvivo engineered onto the surface of de-epithelialised human amnion in amonolayer, after seeding the construct with a suspension of cultured andviable corneal keratinocytes according to the invention. About 500,000cells are used for the seeding and the cells are allowed to cover thesurface of the construct with cells after further incubation of aboutanother 3 weeks. The engineering with cells occurs after about threeweeks of primary cell culture, and re-seeding may be necessary. Theresulting biological cell-biocomposite construct consisting of collagen,amnion fibers and corneal keratinocytes, consisting of membrane andmonolayer of cells.

The corneal cell preparation according to the invention can be spreadonto a dissolvable contact lens that is applied to the damaged cornea.The contact lens disappears and the cells close the corneal defect.

The corneal cell preparation according to the invention can beadministered topically in eye drops in patients suffering from dry eyesymptoms. Alternatively, the above amnion can be used on its own on thescarred cornea or the construct and the cell preparation according tothe invention can be attached to the inside of a biological orartificial contact lens and then applied to the cornea and covered withan eye pad.

The corneal cell preparation according to the invention is useful inalleviating the pain of dry eye, for the treatment of Steven's JohnsonSyndrome and corneal blindness due to acid and corrosive alkali burns inindustry, corneal ulcers such as recalcitrant neurotrophic, herpetic andimmunologically induced corneal ulceration.

Example 14 Autologous Bone Marrow Cell Association Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention wherein bonemarrow cells are provided under step (d) or (e).

Hip bone marrow is harvested and centrifuged in a ready-to-use devicefor the preparation of a platelet concentrate according to the invention(also called RegenBCT™ (Blood Cell Therapy) of in order to separate redblood cells.

The bone marrow cell preparation is then admixed to the plateletconcentrate according to the invention and applied or injected with anapplicator with addition of CaCl₂ to the injured site of the patients.

The bone marrow cell preparation according to the invention is usefulfor the treatment of ischemic and non-ischemic cardiac disease, bonedefect, cartilage defect.

Example 15 Autologous Schwann Cell Association Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention wherein Schwanncells are provided under step (d) or (e).

Under local anaesthesia, a biopsy is performed either the N. Saphenousof N. SURALIS in the lower extremity. The nerve biopsy is cut into smallblocks and primary cultures are induced in Petri dishes enriched anautologous platelet concentrate composition according to the invention,also called RegenPRP™.

Monolayers are expanded in 3D and the cells are eventually harvested bytrypsin digestion and concentrated in a syringe for local infiltrationof the surgically exposed and damaged spinal cord. The cultivated cellshave been shown to contain myelin.

The Schwann cell preparation according to the invention is useful forthe treatment of peripheral nerve damage, nerve suture and spinal cordinjury.

Example 16 Autologous Human Islet Cell Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention whereinpancreas islet cells are provided under step (d) or (e).

Pancreas islets are harvested by open biopsy and separated byconventional enzymatic digestion and Ficoll or Hypaque separation (Pageet al., 2007, Diba. Vas. Dis. Res., 7-12) in a medium enriched with anautologous platelet concentrate composition according to the invention,also called RegenPRP™.

The pancreas islet cell preparation is then injected as a bolus via theportal vein into the liver.

The pancreas islet cell preparation according to the invention is usefulfor the treatment of type1 diabetes or insulin-dependent diabetes andfor the reversal of hyperglycaemia of diabetes mellitus.

Example 17 Autologous Human Osteoblast Cell Preparation

Example of autologous cell association according to the invention can beprepared by using the process according to the invention whereinosteoblast cells are provided under step (d) or (e).

Cortical punch bone biopsy is derived from the iliac crest or equivalentsite (maxilla) under local anaesthesia. The bone biopsy is placedaseptically in DMEM medium at 4° C., or equivalent transport medium bythose experienced in the art of bone and osteoblast culture ex vivo. Thebone biopsy is then diced and digested in diluted 0% type-1 collagenase(Sigma or Boehringer) at 37° C. for 15 min under laminar flow hood.Alternatively, trypsin digestion (Worthington) may be usedalternatively. Enzymatic digestion is terminated with three washes withan autologous platelet concentrate composition according to theinvention, also called RegenPRP™ at 10% in DMEM at 4° C. The preparationis centrifuged, pelleted and resuspended. The bone fragments are platedon Petri dishes or flasks as explants with air-lifting technology in anautologous platelet concentrate composition according to the invention,also called RegenPRP™. The preparation is cultured at 37° C. withantibiotics, gentamicin and amphotericin-B under a gas flow of 95% airand 5% CO₂. The culture medium is changed three times per week, eachtime spiking DMEM medium with 10% vol. of an autologous plateletconcentrate composition according to the invention. The cell viabilityand morphology are evaluated three times a week to assess cell crawling,apoptosis and 3D dimensional monolayer progression. The formation ofmicrofilament and differentiation is assessed by inverted microscopy(Olympus®). Absence of bacterial and viral contamination is checked.Osteoblasts can be engineered onto human amnion to create cellbiocomposite scaffold and cell monolayer carrier/construct aftermembrane seeding with 100,000 cells as obtained above and allowingmonolayer membrane expansion over 3-4 weeks allowing unique constructionof osteoblast-amnion-membrane construct for use and transfer to cover abone defect or grafted area following non-union of fracture in any site.

The osteoblast cell preparation according to the invention is useful forthe treatment of bone defects, bone grafts or bone disorders.

1-30. (canceled)
 31. A sterilized, vacuum sealed separator tube forpreparing a therapeutic platelet concentrate from whole bloodcomprising: an inlet adapted to introduce whole blood; an anticoagulantcomposition comprising either: (i) a sodium citrate solution; or (ii)anhydrous sodium citrate; and a thixotropic gel selected from apolyester-based gel or a polymer mixture gel, the thixotropic gel beingwater insoluble and chemically inert to blood constituents; wherein theseparator tube is adapted to be centrifuged when at least partiallyfilled with the whole blood, at about 1,500 g up to about 2,000 g forabout 3 to about 10 minutes in a single centrifugation to separate bloodcomponents in the whole blood into at least: (i) the therapeuticplatelet concentrate obtained without supernatant removal and containinggreater than 300 billion platelets per liter and less than about0.6×10¹² red blood cells per liter; and (ii) red blood cells.
 32. Theseparator tube according to claim 31, wherein the anticoagulantcomposition comprises a 0.1 M sodium citrate solution.
 33. The separatortube according to claim 31, wherein the anticoagulant compositioncomprises anhydrous sodium citrate.
 34. The separator tube according toclaim 31, wherein the thixotropic gel is a polyester-based gel.
 35. Theseparator tube according to claim 31, wherein the separator tube iscomprised at least of polyethylene terephthalate (PET).
 36. (canceled)37. (canceled)
 38. The separator tube according to claim 31, wherein thetherapeutic platelet concentrate has a platelet yield of about 90% toabout 99% without supernatant removal. 39-50. (canceled)
 51. Theseparator tube according to claim 31, wherein the therapeutic plateletconcentrate contains at least about 400 billion platelets per literwithout supernatant removal.
 52. The separator tube according to claim31, wherein the therapeutic platelet concentrate contains fibrinogen ata concentration of at least about 3 mg/L without supernatant removal.53. The separator tube according to claim 31, wherein the therapeuticplatelet concentrate contains at least about 2.8×10⁹ white blood cellsper liter without supernatant removal.
 54. The separator tube accordingto claim 31, wherein the therapeutic platelet concentrate furthercomprises platelet derived growth factors, (PDGF), vascular endothelialgrowth factors (VEGF), and epidermal growth factor (EGF) that remainstable for a period of at least 72 hours when stored at room temperaturein the separator tube.
 55. A method of making a therapeutic plateletconcentrate comprising: a) providing a sterilized, vacuum sealedseparator tube comprising: an inlet adapted to introduce whole blood; ananticoagulant composition comprising either: (i) a sodium citratesolution; or (ii) anhydrous sodium citrate; and a thixotropic gelselected from a polyester-based gel or a polymer mixture gel, thethixotropic gel being water insoluble and chemically inert to bloodconstituents; b) introducing whole blood to the separator tube throughthe inlet; and c) centrifuging the separator tube containing whole bloodin b) at about 1,500 g up to about 2,000 g for about 3 to about 10minutes only once, d) optionally removing a portion of the supernatantin the tube, wherein after the centrifuging and resuspension, the wholeblood is separated into at least: (i) a therapeutic platelet concentratewherein the therapeutic platelet concentrate contains greater than 300billion platelets per liter and less than about 0.6×10¹² red blood cellsper liter without supernatant removal; and (ii) red blood cells.
 56. Themethod according to claim 55, wherein the anticoagulant compositioncomprises a 0.1 M sodium citrate solution.
 57. The method according toclaim 55, wherein the anticoagulant composition comprises anhydroussodium citrate.
 58. The method according to claim 55, wherein thethixotropic gel is a polyester-based gel.
 59. The method according toclaim 55, wherein the separator tube is comprised at least ofpolyethylene terephthalate (PET).
 60. The method according to claim 55,wherein the therapeutic platelet concentrate contains at least about 400billion platelets per liter without supernatant removal.
 61. The methodaccording to claim 55, wherein the therapeutic platelet concentratecontains fibrinogen at a concentration of at least about 3 mg/L withoutsupernatant removal.
 62. The method according to claim 55, wherein thetherapeutic platelet concentrate contains at least about 2.8×10⁹ whiteblood cells per liter without supernatant removal.
 63. The methodaccording to claim 55, wherein the therapeutic platelet concentratefurther comprises platelet derived growth factors, (PDGF), vascularendothelial growth factors (VEGF), and epidermal growth factor (EGF)that remain stable for a period of at least 72 hours when stored at roomtemperature in the separator tube.
 64. The method according to claim 55,wherein the separator tube has a useable vacuum of from about 8 mL toabout 10 mL.
 65. The method according to claim 55, wherein the separatortube is centrifuged in c) at about 3,800 revolutions per minute (rpm)for about 3.5 minutes.
 66. The separator tube according to claim 31,wherein the thixotropic gel is present in an amount of from about 1 mLto about 3 mL.
 67. The method according to claim 55, wherein thethixotropic gel is present in an amount of from about 1 mL to about 3mL.
 68. The separator tube according to claim 31, wherein theanticoagulant is present in an amount of about 3.5 mg/mL.
 69. The methodaccording to claim 55, wherein the anticoagulant is present in an amountof about 3.5 mg/mL.
 70. The separator tube according to claim 31,wherein the thixotropic gel is non-toxic and non-mutagenic in accordancewith ISO
 10993. 71. The method according to claim 55, wherein thethixotropic gel is non-toxic and non-mutagenic in accordance with ISO10993.
 71. (canceled)
 72. (canceled)
 73. A sterilized, vacuum sealedseparator tube for preparing a cosmetic or skin repair plateletconcentrate from whole blood comprising: an inlet adapted to introducewhole blood; an anticoagulant composition; and a non-toxic andnon-mutagenic thixotropic gel; wherein the separator tube is adapted tobe centrifuged when at least partially filled with the whole blood, atabout 1,500 g up to about 2,000 g for about 3 to about 10 minutes in asingle centrifugation to separate blood components in the whole bloodinto at least: (i) a cosmetic or skin repair platelet concentrateobtained without supernatant removal and containing greater than about300 billion platelets per liter and less than about 0.6×10¹² red bloodcells per liter; and (ii) red blood cells.